Oligonucleotide Conjugates

ABSTRACT

The invention relates to the field of oligonucleotide therapeutics, and in particular to the use of a cleavable, e.g. a phosphodiester region covalently attached to a conjugate, a targeting group or blocking group to enhance the properties of the oligonucleotides, for example to improve the therapeutic index.

FIELD OF INVENTION

The invention relates to the field of oligonucleotide therapeutics, andin particular to the use of a conjugate, a targeting group or blockinggroup to enhance the properties of the oligonucleotides, for example toimprove the therapeutic index.

RELATED CASES

This application claims priority from EP12192773.5, EP13153296.2,EP13157237.2 and EP13174092.0, which are hereby incorporated byreference.

BACKGROUND

Oligonucleotide conjugates have been extensively evaluated for use insiRNAs, where they are considered essential in order to obtainsufficient in vivo potency. For example, see WO2004/044141 refers tomodified oligomeric compounds that modulate gene expression via an RNAinterference pathway. The oligomeric compounds include one or moreconjugate moieties that can modify or enhance the pharmacokinetic andpharmacodynamic properties of the attached oligomeric compound.

In contrast, single stranded antisense oligonucleotides are typicallyadministered therapeutically without conjugation or formulation. Themain target tissues for antisense oligonucleotides are the liver and thekidney, although a wide range of other tissues are also accessible bythe antisense modality, including lymph node, spleen, bone marrow.

WO2008/113832 discloses LNA phosphorothioate gapmer oligonucleotideswhere the flanking regions comprise at least one phosphodiester betweenor adjacent to a LNA nucleoside. The oligomers were preferentiallytargeted to the kidney.

WO2004/087931 refers to oligonucleotides comprising an acid cleavablehydrophilic polymer (PEG) conjugate.

WO 2005/086775 refers to targeted delivery of therapeutic agents tospecific organs using a therapeutic chemical moiety, a cleavable linkerand a labeling domain. The cleavable linker may be, for example, adisulfide group, a peptide or a restriction enzyme cleavableoligonucleotide domain.

WO 2009/126933 refers to specific delivery of siRNA nucleic acids bycombining targeting ligands with endosomolytic components.

WO 2011/126937 refers to targeted intracellular delivery ofoligonucleotides via conjugation with small molecule ligands.

WO2009/025669 refers to polymeric (polyethylene glycol) linkerscontaining pyridyl disulphide moieties. See also Zhao et al.,Bioconjugate Chem. 2005 16 758-766.

Chaltin et al., Bioconjugate Chem. 2005 16 827-836 reports oncholesterol modified mono- di- and tetrameric oligonucleotides used toincorporate antisense oligonucleotides into cationic liposomes, toproduce a dendrimeric delivery system. Cholesterol is conjugated to theoligonucleotides via a lysine linker.

Other non-cleavable cholesterol conjugates have been used to targetsiRNAs and antagomirs to the liver—see for example, Soutscheck et al.,Nature 2004 vol. 432 173-178 and Krutzfeldt et al., Nature 2005 vol 438,685-689. For the partially phosphorothiolated siRNAs and antagomirs, theuse of cholesterol as a liver targeting entity was found to be essentialfor in vivo activity.

The present invention is based upon the discovery that highly effectivetargeted delivery of oligonucleotides is achieved by the use of a homingdevice linked to the oligonucleotide by means of a short region ofnuclease labile nucleosides, such as phosphodiester linked DNA or RNAnucleosides.

SUMMARY OF INVENTION

The invention provides for an oligomeric compound comprising threeregions:

-   -   i) a first region (region A), which comprises 7-26 contiguous        nucleotides;    -   ii) a second region (region B) which comprises between 1-10        nucleotides, which is covalently linked to the 5′ or 3′        nucleotide of the first region, such as via a internucleoside        linkage group such as a phosphodiester linkage, wherein either        -   a. the internucleoside linkage between the first and second            region is a phosphodiester linkage and the nucleoside of the            second region [such as immediately] adjacent to the first            region is either DNA or RNA; and/or        -   b. at least 1 nucleoside of the second region is a            phosphodiester linked DNA or RNA nucleoside;    -   iii) a third region (C) which comprises a conjugate moiety, a        targeting moiety, a reactive group, an activation group, or a        blocking moiety, wherein the third region is covalent linked to        the second region.

In some embodiments, region A and region B form a single contiguousnucleotide sequence of 8-35 nucleotides in length.

In some aspects the internucleoside linkage between the first and secondregions may be considered part of the second region.

In some embodiments, there is a phosphorus containing linkage groupbetween the second and third region. The phosphorus linkage group, may,for example, be a phosphate (phosphodiester), a phosphorothioate, aphosphorodithioate or a boranophosphate group. In some embodiments, thisphosphorus containing linkage group is positioned between the secondregion and a linker region which is attached to the third region. Insome embodiments, the phosphate group is a phosphodiester.

Therefore, in some aspects the oligomeric compound comprises at leasttwo phosphodiester groups, wherein at least one is as according to theabove statement of invention, and the other is positioned between thesecond and third regions, optionally between a linker group and thesecond region.

In some embodiments, the third region is an activation group, such as anactivation group for use in conjugation. In this respect, the inventionalso provides activated oligomers comprising region A and B and anactivation group, e.g. an intermediate which is suitable for subsequentlinking to the third region, such as suitable for conjugation.

In some embodiments, the third region is a reactive group, such as areactive group for use in conjugation. In this respect, the inventionalso provides oligomers comprising region A and B and a reactive group,e.g. an intermediate which is suitable for subsequent linking to thethird region, such as suitable for conjugation. The reactive group may,in some embodiments comprise an amine of alcohol group, such as an aminegroup.

In some embodiments region A comprises at least one, such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 internucleoside linkages other than phosphodiester, such asinternucleoside linkages which are (optionally independently] selectedfrom the group consisting of phosphorothioate, phosphorodithioate, andboranophosphate, and methylphosphonate, such as phosphorothioate. Insome embodiments region A comprises at least one phosphorothioatelinkage. In some embodiments at least 50%, such as at least 75%, such asat least 90% of the internucleoside linkages, such as all theinternucleoside linkages within region A are other than phosphodiester,for example are phosphorothioate linkages. In some embodiments, all theinternucleoside linkages in region A are other than phosphodiester.

In some embodiments, the oligomeric compound comprises an antisenseoligonucleotide, such as an antisense oligonucleotide conjugate. Theantisense oligonucleotide may be or may comprise the first region, andoptionally the second region. In this respect, in some embodiments,region B may form part of a contiguous nucleobase sequence which iscomplementary to the (nucleic acid) target. In other embodiments, regionB may lack complementarity to the target.

Alternatively stated, in some embodiments, the invention provides anon-phosphodiester linked, such as a phosphorothioate linked,oligonucleotide (e.g. an antisense oligonucleotide) which has at leastone terminal (5′ and/or 3′) DNA or RNA nucleoside linked to the adjacentnucleoside of the oligonucleotide via a phosphodiester linkage, whereinthe terminal DNA or RNA nucleoside is further covalently linked to aconjugate moiety, a targeting moiety or a blocking moiety, optionallyvia a linker moiety.

The invention provides for a pharmaceutical composition comprising theoligomeric compound of the invention and a pharmaceutically acceptablediluent, carrier, salt or adjuvant.

The invention provides for the oligomeric compound according to theinvention for use in the inhibition of a nucleic acid target in a cell.In some embodiments the use is in vitro. In some embodiments the use isin vivo.

The invention provides for the oligomeric compound of the invention foruse in medicine, such as for use as a medicament.

The invention provides for the oligomeric compound of the invention foruse in the treatment of a medical disease or disorder.

The invention provides for the use of the oligomeric compound of theinvention for the preparation of a medicament for the treatment of adisease or disorder, such as a metabolic disease or disorder.

The invention provides for a method of synthesizing (or manufacture) ofan oligomeric compound, such as the oligomeric compound of theinvention, said method comprising either:

-   -   a) a step of providing a [solid phase] oligonucleotide synthesis        support to which one of the following is attached [third        region]:        -   i) optionally a linker group (—Y—)        -   ii) a group X comprising a group selected from the group            consisting of a conjugate, a targeting group, a blocking            group, a reactive group [e.g. an amine or an alcohol] or an            activation group (X—), or an an —Y—X group    -   and    -   b) a step of [sequential] oligonucleotide synthesis of region B        followed by region A, and/or:    -   c) a step of [sequential] oligonucleotide synthesis of a first        region (A) and a second region (B), wherein the synthesis step        is followed by    -   d) a step of adding a third region [phosphoramidite comprising]        -   i) optionally a linker group (—Y—)        -   ii) a group X comprising a group selected from the group            consisting of a conjugate, a targeting group, a blocking            group, a reactive group [e.g. an amine or an alcohol] or an            activation group (X—) or optionally an —Y—X group            followed by    -   e) the cleavage of the oligomeric compound from the [solid        phase] support        wherein, optionally said method further comprises a further step        selected from:    -   f) wherein the third group is an activation group, the step of        activating the activation group to produce a reactive group,        followed by adding a conjugate, a blocking, or targeting group        to the reactive group, optionally via a linker group (Y);    -   g) wherein the third region is a reactive group, the step of        adding a conjugate, a blocking, or targeting group to the        reactive group, optionally via a linker group (Y).    -   h) wherein the third region is a linker group (Y), the step of        adding a conjugate, a blocking, or targeting group to the linker        group (Y)        wherein steps f), g) or h) are performed either prior to or        subsequent to cleavage of the oligomeric compound from the        oligonucleotide synthesis support. In some embodiments, the        method may be performed using standard phosphoramidite        chemistry, and as such the region X and/or region X or region X        and Y may be provided, prior to incorporation into the oligomer,        as a phosphoramidite. Please see FIGS. 5-10 which illustrate        non-limiting aspects of the method of the invention.

The invention provides for a method of synthesizing (or manufacture) ofan oligomeric compound, such as the oligomeric compound of theinvention, said method comprising a step of [sequential] oligonucleotidesynthesis of a first region (A) and optionally a second region (B),wherein the synthesis step is followed by a step of adding a thirdregion [phosphoramidite comprising] region X (also referred to as regionC), or Y such as a region comprising a group selected from the groupconsisting of a conjugate, a targeting group, a blocking group, afunctional group, a reactive group [e.g. an amine or an alcohol] or anactivation group (X), or an —Y—X group followed by the cleavage of theoligomeric compound from the [solid phase] support.

It is however recognized that the region X or X—Y may be added after thecleavage from the solid support. Alternatively, the method of synthesismay comprise the steps of synthesizing a first (A), and optionallysecond region (B), followed by the cleavage of the oligomer from thesupport, with a subsequent step of adding a third region, such as X orX—Y group to the oligomer. The addition of the third region may beachieved, by example, by adding an amino phosphoramidite unit in thefinal step of oligomer synthesis (on the support), which can, aftercleavage from the support, be used to join to the X or X—Y group,optionally via an activation group on the X or Y (when present) group.In the embodiments where the cleavable linker is not a nucleotideregion, region B may be a non-nucleotide cleavable linker for example apeptide linker, which may form part of region X (also referred to asregion C) or be region Y (or part thereof).

In some embodiments of the method, region X (such as C) or (X—Y), suchas the conjugate (e.g. a GalNAc conjugate) comprises an activationgroup, (an activated functional group) and in the method of synthesisthe activated conjugate (or region x, or X—Y) is added to the first andsecond regions, such as an amino linked oligomer. The amino group may beadded to the oligomer by standard phosphoramidite chemistry, for exampleas the final step of oligomer synthesis (which typically will result inamino group at the 5′ end of the oligomer). For example during the laststep of the oligonucleotide synthesis a protected amino-alkylphosphoramidite is used, for example a TFA-aminoC6 phosphoramidite(6-(Trifluoroacetylamino)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite).

Region X (or region C as referred to herein), such as the conjugate(e.g. a GalNac conjugate) may be activated via NHS ester method and thenthe aminolinked oligomer is added. For example a N-hydroxysuccinimide(NHS) may be used as activating group for region X (or region C, such asa conjugate, such as a GalNac conjugate moiety.

The invention provides an oligomer prepared by the method of theinvention.

In some embodiments, region X and/or region X or region X and Y may becovalently joined (linked) to region B via a phosphate nucleosidelinkage, such as those described herein, including phosphodiester orphosphorothioate, or via an alternative group, such as a triazol group.

The invention provides for a method of treatment of a disease ordisorder in a subject in need of treatment, said method comprising thesteps of administering a pharmaceutical composition comprising theoligomeric compound of the invention to said subject in atherapeutically effective amount.

The invention provides for a method of inhibiting the expression of atarget gene in a cell, said method comprising administering theoligomeric compound according to the invention to a cell which isexpressing said target gene, suitably in an amount effective to reducethe expression of the target gene in said cell. In some embodiments themethod is in vitro (.e. not in an organism, but may be in a (e.g.ex-vivo) cell or tissue). In some embodiments the method is in vivo.

The invention also provides for an LNA oligomer, comprising a contiguousregion of 8-24 phosphorothioate linked nucleosides, and furthercomprising between 1 and 6 DNA nucleosides which are contiguous with theLNA oligomer, wherein the internucleoside linkages between the DNA,and/or adjacent to the DNA nucleoside(s), is physiologically labile,such as is/are phosphodiester linkages. Such an LNA oligomer may be inthe form of a conjugate, as described herein, or may, for example be anintermediate to be used in a subsequent conjugation step. Whenconjugated, the conjugate may, for example be or comprise a sterol, suchas cholesterol or tocopherol, or may be or comprise a (non-nucleotide)carbohydrate, such as a GalNac conjugate, such as a GalNac cluster, e.g.triGalNac, or another conjugate as described herein.

The invention provides for an LNA antisense oligomer (which may bereferred to as region A herein) comprising an antisense oligomer and anasialoglycoprotein receptor targeting moiety conjugate moiety, such as aGalNAc moiety, which may form part of a further region (referred to asregion C). The LNA antisense oligomer may be 7-30, such as 8-26nucleosides in length and it comprises at least one LNA unit(nucleoside).

The invention provides for an LNA antisense oligomer covalently joinedto (e.g. linked to) a (non-nucleoside) carbohydrate moiety, such as acarbohydrate conjugate moiety. In some embodiments the carbohydratemoiety is not a linear carbohydrate polymer. The carbohydrate moiety mayhowever be multi-valent, such as, for example 2, 3, 4 or 4 identical ornon-identical carbohydrate moieties may be covalently joined to theoligomer, optionally via a linker or linkers.

The invention provides for an LNA antisense oligomer (conjugate)comprising an antisense oligomer and a conjugate moiety which comprisesa carbohydrate, such as a carbohydrate conjugate moiety.

The invention provides for a pharmaceutical composition comprising theLNA oligomeric compound of the invention and a pharmaceuticallyacceptable diluent, carrier, salt or adjuvant.

The invention provides for the oligomeric compound according to theinvention for use in the inhibition of a nucleic acid target in a cell.In some embodiments the use is in vitro. In some embodiments the use isin vivo.

The invention provides for the oligomeric compound of the invention foruse in medicine, such as for use as a medicament.

The invention provides for the oligomeric compound of the invention foruse in the treatment of a medical disease or disorder.

The invention provides for the use of the oligomeric compound of theinvention for the preparation of a medicament for the treatment of adisease or disorder, such as a metabolic disease or disorder.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Non-limiting illustration of oligomers of the invention attachedto an activation group (i.e. a protected reactive group—as the thirdregion). The internucleoside linkage L may be, for examplephosphodiester, phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, such as phosphodiester. PO is a phosphodiesterlinkage. Compound a) has a region B with a single DNA or RNA, thelinkage between the second and the first region is PO. Compound b) hastwo DNA/RNA (such as DNA) nucleosides linked by a phosphodiesterlinkage. Compound c) has three DNA/RNA (such as DNA) nucleosides linkedby a phosphodiester linkages. In some embodiments, Region B may befurther extended by further phosphodiester DNA/RNA (such as DNAnucleosides). The activation group is illustrated on the left side ofeach compound, and may, optionally be linked to the terminal nucleosideof region B via a phosphorus nucleoside linkage group, such asphosphodiester, phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or in some embodiments a triazole linkage. Compoundsd), e), & f) further comprise a linker (Y) between region B and theactivation group, and region Y may be linked to region B via, forexample, a phosphorus nucleoside linkage group, such as phosphodiester,phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or in some embodiments a triazole linkage.

FIG. 2: Equivalent compounds as shown in FIG. 1; however a reactivegroup is used in place of the activation group. The reactive group may,in some embodiments be the result of activation of the activation group(e.g. deprotection). The reactive group may, in non-limiting examples,be an amine or alcohol.

FIG. 3: Non-limiting Illustration of compounds of the invention. Samenomenclature as FIG. 1. X may in some embodiments be a conjugate, suchas a lipophilic conjugate such as cholesterol, or another conjugate suchas those described herein. In addition, or alternatively X may be atargeting group or a blocking group. In some aspects X may be anactivation group (see FIG. 1), or a reactive group (see FIG. 2). X maybe covalently attached to region B via a phosphorus nucleoside linkagegroup, such as phosphodiester, phosphorothioate, phosphorodithioate,boranophosphate or methylphosphonate, or may be linked via analternative linkage, e.g. a triazol linkage (see L in compounds d), e),and f)).

FIG. 4. Non-limiting Illustration of compounds of the invention, wherethe compounds comprise the optional linker between the third region (X)and the second region (region B). Same nomenclature as FIG. 1. Suitablelinkers are disclosed herein, and include, for example alkyl linkers,for example C6 linkers. In compounds A, B and C, the linker between Xand region B is attached to region B via a phosphorus nucleoside linkagegroup, such as phosphodiester, phosphorothioate, phosphorodithioate,boranophosphate or methylphosphonate, or may be linked via analternative linkage e.g. a triazol linkage (Li). In these compounds Liirepresents the internucleoside linkage between the first (A) and secondregions (B).

FIGS. 5a and b . 5 b shows a non-limiting example of a method ofsynthesis of compounds of the invention. US represent a oligonucleotidesynthesis support, which may be a solid support. X is the third region,such as a conjugate, a targeting group, a blocking group etc. In anoptional pre-step, X is added to the oligonucleotide synthesis support.Otherwise the support with X already attached may be obtained (i). In afirst step, region B is synthesized (ii), followed by region A (iii),and subsequently the cleavage of the oligomeric compound of theinvention from the oligonucleotide synthesis support (iv). In analternative method the pre-step involves the provision of aoligonucleotide synthesis support with a region X and a linker group (Y)attached (see FIG. 5a ). In some embodiments, either X or Y (if present)is attached to region B via a phosphorus nucleoside linkage group, suchas phosphodiester, phosphorothioate, phosphorodithioate, boranophosphateor methylphosphonate, or an alternative linkage, such as a triazollinkage.

FIG. 6. A non-limiting example of a method of synthesis of compounds ofthe invention which comprise a linker (Y) between the third region (X)and the second region (B). US represents a oligonucleotide synthesissupport, which may be a solid support. X is the third region, such as aconjugate, a targeting group, a blocking group etc. In an optionalpre-step, Y is added to the oligonucleotide synthesis support. Otherwisethe support with Y already attached may be obtained (i). In a firststep, region B is synthesized (ii), followed by region A (iii), andsubsequently the cleavage of the oligomeric compound of the inventionfrom the oligonucleotide synthesis support (iv). In some embodiments (asshown), region X may be added to the linker (Y) after the cleavage step(v). In some embodiments, Y is attached to region B via a phosphorusnucleoside linkage group, such as phosphodiester, phosphorothioate,phosphorodithioate, boranophosphate or methylphosphonate, or analternative linkage, such as a triazol linkage.

FIG. 7. A non-limiting example of a method of synthesis of compounds ofthe invention which utilize an activation group. In an optionalpre-step, the activation group is attached the oligonucleotide synthesissupport (i), or the oligonucleotide synthesis support with activationgroup is otherwise obtained. In step ii) region B is synthesizedfollowed by region A (iii). The oligomer is then cleaved from theoligonucleotide synthesis support (iv). The intermediate oligomer(comprising an activation group) may then be activated (vl) or (viii)and a third region (X) added (vi), optionally via a linker (Y) (ix). Insome embodiments, X (or Y when present) is attached to region B via aphosphorus nucleoside linkage group, such as phosphodiester,phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or an alternative linkage, such as a triazol linkage.

FIG. 8. A non-limiting example of a method of synthesis of compounds ofthe invention, wherein a bifunctional oligonucleotide synthesis supportis used (i). In such a method, either the oligonucleotide is synthesizedin an initial series of steps (ii)-(iii), followed by the attachment ofthe third region (optionally via a linker group Y), the oligomericcompound of the invention may then be cleaved (v). Alternatively, asshown in steps (vi)-(ix), the third region (optionally with a linkergroup (Y) is attached to the oligonucleotide synthesis support (this maybe an optional pre-step)—or an oligonucleotide synthesis support withthe third region (optionally with Y) is otherwise provided, theoligonucleotide is then synthesized (vii-viii). The oligomeric compoundof the invention may then be cleaved (ix). In some embodiments, X (or Ywhen present) is attached to region B via a phosphorus nucleosidelinkage group, such as phosphodiester, phosphorothioate,phosphorodithioate, boranophosphate or methylphosphonate, or analternative linkage, such as a triazol linkage. The US may in someembodiment, prior to the method (such as the pre-step) comprise a stepof adding a bidirectional (bifunctional) group which allows theindependent synthesis of the oligonucleotide and the covalent attachmentof group X, Y (or X and Y) to support (as shown)—this may for example beachieved using a triazol or of nucleoside group. The bidirectional(bifunctional) group, with the oligomer attached, may then be cleavedfrom the support.

FIG. 9. A non-limiting example of a method of synthesis of compounds ofthe invention: In an initial step, the first region (A) is synthesized(ii), followed by region B. In some embodiments the third region is thenattached to region B (iii), optionally via a phosphate nucleosidelinkage (or e.g. a trialzol linkage). The oligomeric compound of theinvention may then be cleaved (iv). When a linker (Y) is used, in someembodiments the steps (v)-(viii) may be followed: after synthesis ofregion B, the linker group (Y) is added, and then either attached to (Y)or in a subsequent step, region X is added (vi). The oligomeric compoundof the invention may then be cleaved (vii). In some embodiments, X (or Ywhen present) is attached to region B via a phosphorus nucleosidelinkage group, such as phosphodiester, phosphorothioate,phosphorodithioate, boranophosphate or methylphosphonate, or analternative linkage, such as a triazol linkage.

FIG. 10. A non-limiting example of a method of synthesis of compounds ofthe invention: In this method an activation group is used: Steps(i)-(iii) are as per FIG. 9. However after the oligonucleotide synthesis(step iii), an activation group (or a reactive group) is added to regionB, optionally via a phosphate nucleoside linkage. The oligonucleotide isthen cleaved from the support (v). The activation group may besubsequently activated to produce a reactive group, and then the thirdregion (X), such as the conjugate, blocking group or targeting group, isadded to the reactive group (which may be the activated activation groupor the reactive group), to produce the oligomer (vi). As shown in(vii)-(viii), after cleavage, a linker group (Y) is added (vii), andthen either attached to (Y) or in a subsequent step, region X is addedto produce the oligomer (viii). It should be recognized that in analternative all of the steps (ii)-(viii) may be performed on theoligonucleotide synthesis support, and in such instances a final step ofcleaving the oligomer from the support may be performed. In someembodiments, the reactive group or activation group is attached toregion B via a phosphorus nucleoside linkage group, such asphosphodiester, phosphorothioate, phosphorodithioate, boranophosphate ormethylphosphonate, or an alternative linkage, such as a triazol linkage.

FIG. 11. Silencing of ApoB mRNA with Cholesterol-conjugates in vivo.Mice were injected with a single dose of 1 mg/kg unconjugatedLNA-antisense oligonucleotide (#3833) or equimolar amounts of LNAantisense oligonucleotides conjugated to Cholesterol with differentlinkers (Tab. 3) and sacrificed at days 1, 3, 7 and 10 after dosing. RNAwas isolated from liver and kidney and subjected to ApoB specificRT-qPCR A. Quantification of ApoB mRNA from liver samples normalized toGAPDH and shown as percentage of the average of equivalent salinecontrols B. Quantification of ApoB mRNA from kidney samples normalizedto GAPDH and shown as percentage of the average of equivalent salinecontrols.

FIG. 12. Shows the cholesterol C6 conjugate which may be used as X—Y— incompounds of the invention, as well as specific compounds used in theexamples, include specific compounds of the invention.

FIG. 13. Examples of cholesterol, trivalent GalNac, FAM, folic acid,monovalent GalNac and tocopherol conjugates used in the experiments(e.g. compounds of FIG. 12).

FIG. 14. Silencing of ApoB mRNA with cholesterol-conjugates in vivo.Mice were injected with a single dose of 1 mg/kg unconjugatedLNA-antisense oligonucleotide (#3833) or equimolar amounts of LNAantisense oligonucleotides conjugated to Cholesterol with differentlinkers (Tab. 3) and sacrificed at days 1, 3, 7, 10, 13 and 16 afterdosing. RNA was isolated from liver and kidney and subjected to ApoBspecific RT-qPCR A. Quantification of ApoB mRNA from liver samplesnormalized to GAPDH and shown as percentage of the average of equivalentsaline controls B. Quantification of ApoB mRNA from kidney samplesnormalized to GAPDH and shown as percentage of the average of equivalentsaline controls.

FIG. 15. Content of the specific LNA oligonucleotide in liver and kidneyin vivo. Mice were injected with a single dose of 1 mg/kg unconjugatedLNA-antisense oligonucleotide (#1) or equimolar amounts of LNA antisenseoligonucleotides conjugated to Cholesterol with different linkers (Tab.4) and sacrificed at days 1, 3, 7, 10, 13 and 16 after dosing. LNAoligonucleotide content was measured using LNA based sandwich ELISAmethod.

FIG. 16. Silencing of PCSK9 mRNA with cholesterol-conjugates in vivo.Mice were injected with a single dose of 10 mg/kg unconjugatedLNA-antisense oligonucleotide (#7) or equimolar amounts of LNA antisenseoligonucleotides conjugated to Cholesterol with different linkers (Tab.5) and sacrificed at days 1, 3, 7 and 10 after dosing. RNA was isolatedfrom liver and kidney and subjected to PCSK9 specific RT-qPCR A.Quantification of PCSK9 mRNA from liver samples normalized to BACT andshown as percentage of the average of equivalent saline controls B.Quantification of PCSK9 mRNA from kidney samples normalized to BACT andshown as percentage of the average of equivalent saline controls.

FIG. 17 Examples of tri-GalNac conjugates which may be used. Conjugates1-4 illustrate 4 suitable GalNac conjugate moieties, and conjugates1a-4a refer to the same conjugates with an additional linker moiety (Y)which is used to link the conjugate to the oligomer (region A or to abiocleavable linker, such as region B). The wavy line represents thecovalent link to the oligomer. Also shown are examples of cholesteroland tocopherol conjugate moieties (5a and 6a). The wavy line representsthe covalent link to the oligomer.

FIG. 18: Example 7a: FVII serum protein levels

FIG. 19: Example 7a: FVII mRNA levels in liver day 4

FIG. 20: Example 7a: Oligonucleotide content in liver and kidney day 4

FIG. 21: Example 7b—FVII serum protein levels

FIG. 22: FVII mRNA levels in liver day 24

FIG. 23: Oligonucleotide content in liver and kidney day 4

FIG. 24. In vivo silencing of ApoB mRNA with different conjugates andPO-linker.

Mice were treated with 1 mg/kg of ASO with different conjugates eitherwithout biocleavable linker, with Dithio-linker (SS) or withDNA/PO-linker (PO). RNA was isolated from liver (A) and kidney samples(B) and analyzed for ApoB mRNA knock down. Data is shown compared toSaline (=1).

FIG. 25. In vitro silencing of Target X mRNA with looped LNA ASO withPO-linker.

Neuro 2a cells were treated with looped LNA ASOs with or withoutPO-linker, respectively. After 6 days gymnosis mRNA was extracted andanalyzed for target X mRNA knock down. mRNA expression is shown aspercentage of mock treated samples.

FIG. 26. Further examples of oligomers depicted in FIG. 1.

DESCRIPTION OF THE INVENTION

The invention relates to oligomeric compounds, such as antisenseoligonucleotides, which are covalently linked to a conjugate group, atargeting group, a reactive group, an activation group, or a blockinggroup, via a short region comprising (e.g. 1-10) of phosphodiesterlinked DNA or RNA nucleoside(s).

The Oligomer

The present invention employs oligomeric compounds (also referred hereinas oligomers) for use in modulating, such as inhibiting a target nucleicacid in a cell. The oligomers may have a length of 8-35 contiguousnucleotides and comprise a first region of 7-25 contiguous nucleotides,and a second region of 1-10 contiguous nucleotides, wherein, forexample, either the internucleoside linkage between the first and secondregion is a phosphodiester linked to the first (or only) DNA or RNAnucleoside of the second region, or region B comprises at least onephosphodiester linked DNA or RNA nucleoside.

The second region may, in some embodiments, comprise further DNA or RNAnucleosides which may be phosphodiester linked. The second region isfurther covalently linked to a third region which may, for example, be aconjugate, a targeting group a reactive group, and/or a blocking group.

In some aspects, the present invention is based upon the provision of alabile region, the second region, linking the first region, e.g. anantisense oligonucleotide, and a conjugate or functional group, e.g. atargeting or blocking group. The labile region comprises at least onephosphodiester linked nucleoside, such as a DNA or RNA nucleoside, suchas 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphodiester linked nucleosides,such as DNA or RNA. In some embodiments, the oligomeric compoundcomprises a cleavable (labile) linker. In this respect the cleavablelinker is preferably present in region B (or in some embodiments,between region A and B).

The term “oligomer” in the context of the present invention, refers to amolecule formed by covalent linkage of two or more nucleotides (i.e. anoligonucleotide). Herein, a single nucleotide (unit) may also bereferred to as a monomer or unit. In some embodiments, the terms“nucleoside”, “nucleotide”, “unit” and “monomer” are usedinterchangeably. It will be recognized that when referring to a sequenceof nucleotides or monomers, what is referred to is the sequence ofbases, such as A, T, G, C or U.

The oligomer consists or comprises of a contiguous nucleotide sequenceof from 8-25, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 nucleotides in length, such as 10-20 nucleotides inlength.

In various embodiments, the compound of the invention does not compriseRNA (units). In some embodiments, the compound according to theinvention, the first region, or the first and second regions together(e.g. as a single contiguous sequence), is a linear molecule or issynthesized as a linear molecule. The oligomer may therefore be singlestranded molecule. In some embodiments, the oligomer does not compriseshort regions of, for example, at least 3, 4 or 5 contiguousnucleotides, which are complementary to equivalent regions within thesame oligomer (i.e. duplexes). The oligomer, in some embodiments, may benot (essentially) double stranded. In some embodiments, the oligomer isessentially not double stranded, such as is not a siRNA.

In some embodiments, the oligomer may comprise a first region which doesnot comprise short regions of, for example, at least 3, 4 or 5contiguous nucleotides, which are complementary to regions within thesame first region (i.e. intra-region duplexes). In this respect, thefirst oligomer may, in some embodiments not form a hybridization with anon-covalently linked complementary strand, e.g. does not form part ofan siRNA.

For example, in some embodiments, the oligomeric compound does comprisea region of complementarity, e.g. when the first region forms part of ansiRNA, or for example, when the third region comprises an aptamer or ablocking oligonucleotide, the oligomeric compound of the invention may,in some embodiments comprise regions of double stranded nucleic acid. Insuch embodiments, regions of double stranded nucleic acid, for exampleforming a duplex of at least 3, such as at least, 4, such as at least 5,such as at least 6 nucleotides in length, may be within the thirdregion, or between the third region and for example the first region, orin some embodiments, the second region, or a region across the first andsecond regions (e.g. when the third region comprises a oligonucleotideblocking region).

In some embodiments, the oligomeric compound is not in the form of aduplex with a (substantially) complementary oligonucleotide—e.g. is notan siRNA.

In some embodiments, the oligomeric compound is a LNA oligomer, forexample an LNA antisense oligomer, (which may be referred to as region Aherein) comprising an antisense oligomer, region B as defined herein,and a carbohydrate conjugate (which may be referred to as region C). TheLNA antisense oligomer may be 7-30, such as 8-26 nucleosides in lengthand it comprises at least one LNA unit (nucleoside). In some embodimentsthe carbohydrate moiety is not a linear carbohydrate polymer.

In some embodiments, the oligomeric compound is a LNA oligomer, forexample an LNA antisense oligomer, (which may be referred to as region Aherein) comprising an antisense oligomer, region B as defined herein,and an asialoglycoprotein receptor targeting moiety conjugate moiety,such as a GalNAc moiety (which may be referred to as region C). Thecarbohydrate moiety may be multi-valent, such as, for example 2, 3, 4 or4 identical or non-identical carbohydrate moieties may be covalentlyjoined to the oligomer, optionally via a linker or linkers (such asregion Y).

The First Region

In some embodiments, the first region may comprise a nucleic acid basedoligomer, such as an antisense oligonucleotide. In some embodiments, thefirst region comprises or consists of a phosphorothioate linkedoligonucleotide, such as an antisense oligonucleotide, of 7-25nucleotides in length. The first region may comprise at least onemodified nucleoside (a nucleoside analogue), such as at least onebicyclic nucleoside (e.g. LNA) or 2′substituted nucleoside. In someembodiments, some or all of the nucleosides of the first region may bemodified nucleosides, also referred to as nucleoside analogues herein.In some embodiments, the modified nucleosides are sugar-modified (e.g.comprise a sugar or sugar surrogate moiety other than ribose ordeoxyribose).

In some embodiments, the first region is an antisense oligomer(antisense oligonucleotide), such as a single stranded oligomer whichcomprises a sequence which is complementary to a nucleic acid target.

In some embodiments the first region comprises or is a gapmer. In someembodiments the first region comprises or is a mixmer. In someembodiments the first region comprises or is a totalmer.

In some embodiments, the first region comprises at least one, such as atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, at least 20, at least 21, at least 22, at least 23, at least24 or 25 nucleoside analogues. In some embodiments the nucleosideanalogues are (optionally independently selected from the groupconsisting of bicyclic nucleoside analogues (such as LNA), and/or 2′substituted nucleoside analogues, such as (optionally independently)selected from the group consisting of 2′-O-alkyl-RNA units, 2′-OMe-RNAunits, 2′-amino-DNA units, 2′-AP, 2′-FANA, 2′-(3-hydroxy)propyl, and2′-fluoro-DNA units, and/or other (optionally) sugar modified nucleosideanalogues such as morpholino, peptide nucleic acid (PNA), CeNA, unlinkednucleic acid (UNA), hexitol nucleoic acid (HNA). bicyclo-HNA (see e.g.WO2009/100320), In some embodiments, the nucleoside analogues increasethe affinity of the first region for its target nucleic acid (or acomplementary DNA or RNA sequence). Various nucleoside analogues aredisclosed in Freier & Altmann; Nucl. Add Res., 1997, 25, 4429-4443 andUhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, herebyincorporated by reference.

In some embodiments, the oligomer, such as the first region thereof,such as the gapmer, mixmer or totalmer comprise at least one bicyclicnucleotide analogue, such as LNA. In some embodiments, the first regioncomprises of at least one bicyclic nucleoside analogues (e.g. LNA)and/or 2′substituted nucleoside analogues. In some embodiments, thenucleoside analogues present in the first region all comprise the samesugar modification. In some embodiments, at least one nucleosideanalogue present in the first region is a bicyclic nucleoside analogue,such as at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, for example allnucleoside analogues (or in a totalmer all nucleosides) bicyclicnucleoside analogues, such as LNA, e.g. beta-D-X-LNA or alpha-L-X-LNA(wherein X is oxy, amino or thio), or other LNAs disclosed hereinincluding, but not limited to, (R/S) cET, cMOE or 5′-Me-LNA. In someembodiments, the oligomer, or first region thereof, comprises of DNA andsugar modified nucleoside analogues, such as bicyclic nucleosideanalogues and/or 2′substituted nucleoside analogues. In someembodiments, the oligomer or first region thereof, comprises of DNA andLNA nucleoside analogues.

WO05013901, WO07/027775, WO07027894 refers to filly 2′substitutedoligomers, such as fully 2′-O-MOE. In some embodiments, the first regionof the oligomer may comprise of 2′ substituted nucleosides. WO07/027775also refers to MOE, LNA, DNA mixmers for use in targeting microRNAs.

In some embodiments, the first region, or the first and second regioncombined to not comprise a region of more than 4 or 5 consecutive DNAunits. Such first regions may be (essentially) unable to recruit RNAseH.

The first region is covalently linked to the second region, such as viaa 5′ terminal or 3′ terminal internucleoside linkage, such as aphosphodiester linkage. The phosphodiester linkage may therefore bepositioned between the 5′ most nucleoside of region A and the 3′ mostnucleoside of region B, and/or between the 3′ most nucleoside of regionA and the 5′ most nucleoside of region B. In this respect, in someembodiments, there may be two region B covalently joined to region A,one at the 5′ terminus of region A and one at the 3′ terminus of regionA. The two region Bs may be the same or different, and they may becovalently linked to the same or different third regions, optionally andindependently via a linker (Y).

In some embodiments, some or all of the nucleosides of the first regionmay be modified nucleosides, also referred to as nucleoside analoguesherein, such as sugar modified nucleoside analogues, for examplebicyclic nucleoside analogues (e.g. LNA) and/or 2′substituted nucleosideanalogues. In some embodiments, the nucleoside analogues present in thefirst region all comprise the same sugar modification, for example areall bicyclic nucleoside analogues, such as LNA, e.g. beta-D-X-LNA oralpha-L-X-LNA (wherein X is oxy, amino orthio), or other LNAs disclosedherein including, but not limited to, (R/S) cET, cMOE or 5′-Me-LNA.

In some embodiments, the internucleoside linkages of the first regioncomprise at least one internucleoside linkage other than phosphodiester,such as at least one, such as at least 50%, such as at least 75%, suchas at least 90%, such as 100% of the internucleoside linkages in regionA are other than phosphodiester. In some embodiments, theinternucleoside linkages other than phosphodiester are sulphurcontaining internucleoside linkages, such as phosphorothioate,phosphorodithioate and boranophosphate, such as phosphorothioate.

The Second Region (Region B)

The second region may comprise or consists of at least one DNA or RNAnucleosides linked to the first region via a phosphodiester linkage. Insome aspects, the internucleoside linkage between the first and secondregion is considered as part of region B.

In some embodiments, the second region comprises or consists of at leastbetween 1 and 10 linked nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9or 10 linked DNA or RNA nucleotides. Whilst a region of DNA/RNAphosphodiester is considered important in the provision of a cleavablelinker, it is possible that region B also comprises sugar-modifiednucleoside analogues, such as those referred to under the first regionabove. However in some embodiments, the nucleosides of region B are(optionally independently) selected from the group consisting of DNA andRNA. It will be recognized that the nucleosides of region B may comprisenaturally occurring or non-naturally occurring nucleobases. Region Bcomprises at least one phosphodiester linked DNA or RNA nucleoside(which may, in some embodiments, be the first nucleoside adjacent toregion A). If region B comprises other nucleosides, region B may alsocomprise of other nucleoside linkages other than phosphodiester, such as(optionally independently) phosphorothioate, phosphodithioate,boranophosphate or methyl phosphonate. However, in other embodiments,all the internucleoside linkages in region B are phosphorothioate. Insome embodiments, all the nucleosides of region B comprise (optionallyindependently) either a 2′-OH ribose sugar (RNA) or a 2′-H sugar—i.e.RNA or DNA.

In some embodiments, the second region comprises or consists of at leastbetween 1 and 10 (e.g. phosphodiester) linked DNA or RNA nucleosides,such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g. phosphodiester) linked DNAor RNA nucleotides.

In some embodiments, region B comprises no more than 3 or no more than 4consecutive DNA or RNA nucleosides (such as DNA nucleosides. As suchregion B may be so short as it does not recruit RNAseH, an aspect whichmay be important when region B does not form a part of a singlecontiguous nucleobase sequence which is complementary to the target.Shorter region Bs, e.g. of 1-4nts in length may also be preferable insome embodiments, as they are unlikely to be the target of sequencespecific restriction enzymes. As such it is possible to vary thesusceptibility of the region B to endonuclease cleavage, and therebyfine-tune the rate of activation of the active oligomer in vivo, or evenintra-cellular. Suitably, if very rapid activation is required, longerregion Bs may be employed and/or region Bs which comprise therecognition sites of (e.g. cell or tissue specific or differentiallyexpressed) restriction enzymes.

As illustrated in the examples, region B may be conjugated to theconjugate, targeting reactive group, an activation group, or blockinggroup (X) via a linker group which may, for example, comprise aphosphodiester linkage, and/or optionally a suitable linker group, suchas those provided herein. For example a phosphate nucleoside linkage(e.g. phosphodiester, phosphorothioate, phosphodithioate,boranophosphate or methylphosphonate) or a triazol group. In someaspects, the linkage group is the same as the linkage group betweenregions A and B, and as such may be a phosphodiester linkage. In someaspects, the linkage group is a phosphorothioate linkage.

In some embodiments the DNA or RNA nucleotides of the second region areindependently selected from DNA and RNA nucleotides. In some embodimentsthe DNA or RNA nucleotides of the second region are DNA nucleotides. Insome embodiments the DNA or RNA nucleotides of the second region are RNAnucleotides.

In the context of the second region, the term DNA and RNA nucleoside maycomprise a naturally occurring or non-naturally occurring base (alsoreferred to as a base analogue or modified base).

It will be recognized that, in some embodiments, the second region mayfurther comprise other nucleotides or nucleotide analogues. In someembodiments, the second region comprises only DNA or RNA nucleosides. Insome embodiments, when the second region comprises more than onenucleoside, the internucleoside linkages in the second region comprisephosphodiester linkages. In some embodiments, when the second regioncomprises more than one nucleoside, all the internucleoside linkages inthe second region comprise phosphodiester linkages.

In some embodiments, at least two consecutive nucleosides of the secondregion are DNA nucleosides (such as at least 3 or 4 or 5 consecutive DNAnucleotides). In some embodiments the at least two consecutivenucleosides of the second region are RNA nucleosides (such as at least 3or 4 or 5 consecutive RNA nucleotides). In some embodiments the at leasttwo consecutive nucleosides of the second region are at least one DNAand at least one RNA nucleoside. The internucleoside linkage betweenregion A and region B is a phosphodiester linkage. In some embodiments,when region B comprises more than one nucleoside, at least one furtherinternucleoside linkage is phosphodiester-such as the linkage group(s)between the 2 (or 3 or 4 or 5) nucleosides adjacent to region A.

The second region is flanked on one side (either 5′ or 3′) by the firstregion, e.g. an antisense oligonucleotide, and on the other side (either3′ or 5′ respectfully, via a conjugate moiety or similar group (e.g. ablocking moiety/group, a targeting moiety/group or therapeutic smallmolecule moiety), optionally via a linker group (i.e. between the secondregion and the conjugate/blocking group etc. moiety).

In such an embodiment, the oligonucleotide of the invention may bedescribed according to the following formula:

5-A-PO—B[Y)X-3′ or 3-A-PO—B[Y)X-5′

wherein A is region A, PO is a phosphodiester linkage, B is region B, Yis an optional linkage group, and X is a conjugate, a targeting, ablocking group or a reactive or activation group.

In some embodiments, region B comprises 3′-5′ or 5′-3′: i) aphosphodiester linkage to the 5′ nucleoside of region A, ii) a DNA orRNA nucleoside, such as a DNA nucleoside, and iii) a furtherphosphodiester linkage

5-A-PO—B—PO-3′ or 3-A-PO—B—PO-5′

The further phosphodiester linkage link the region B nucleoside with oneor more further nucleoside, such as one or more DNA or RNA nucleosides,or may link to X (is a conjugate, a targeting or a blocking group or areactive or activation group) optionally via a linkage group (Y).

In some embodiments, region B comprises 3′-5′ or 5′-3′: i) aphosphodiester linkage to the 5′ nucleoside of region A, ii) between2-10 DNA or RNA phosphodiester linked nucleosides, such as a DNAnucleoside, and optionally iii) a further phosphodiester linkage:

5′-A-[PO—B]n-[Y]—X 3′ or 3′-A-[PO—B]n-[Y]—X 5′

5′-A-[PO—B]n-PO—[Y]—X 3′ or 3′-A-[PO—B]n-PO—[Y]—X 5′

Wherein A represent region A, [PO—B]n represents region B, wherein n is1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, PO is an optionalphosphodiester linkage group between region B and X (or Y if present).

In some embodiments the invention provides compounds according to (orcomprising) one of the following formula:

5′ [Region A]-PO-[region B] 3′-Y—X

5′ [Region A]-PO-[region B]—PO 3′-Y—X

5′ [Region A]-PO-[region B] 3′-X

5′ [Region A]-PO-[region B]—PO 3′-X

3′ [Region A]-PO-[region B] 5′-Y—X

3′ [Region A]-PO-[region B]—PO 5′-Y—X

3′ [Region A]-PO-[region B] 5′-X

3′ [Region A]-PO-[region B]—PO 5′-X

Region B, may for example comprise or consist of:

5′ DNA3′

3′ DNA 5′

5′ DNA-PO-DNA-3′

3′ DNA-PO-DNA-5′

5′ DNA-PO-DNA-PO-DNA 3′

3′ DNA-PO-DNA-PO-DNA 5′

5′ DNA-PO-DNA-PO-DNA-PO-DNA 3′

3′ DNA-PO-DNA-PO-DNA-PO-DNA 5′

5′ DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 3′

3′ DNA-PO-DNA-PO-DNA-PO-DNA-PO-DNA 5′

Sequence Selection in the Second Region:

In some embodiments, region B does not form a complementary sequencewhen the oligonucleotide region A and B is aligned to the complementarytarget sequence.

In some embodiments, region B does form a complementary sequence whenthe oligonucleotide region A and B is aligned to the complementarytarget sequence. In this respect region A and B together may form asingle contiguous sequence which is complementary to the targetsequence.

In some embodiments, the sequence of bases in region B is selected toprovide an optimal endonuclease cleavage site, based upon thepredominant endonuclease cleavage enzymes present in the target tissueor cell or sub-cellular compartment. In this respect, by isolating cellextracts from target tissues and non-target tissues, endonucleasecleavage sequences for use in region B may be selected based upon apreferential cleavage activity in the desired target cell (e.g.liver/hepatocytes) as compared to a non-target cell (e.g. kidney). Inthis respect, the potency of the compound for target down-regulation maybe optimized for the desired tissue/cell.

In some embodiments region B comprises a dinucleotide of sequence AA,AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein Cmay be 5-mthylcytosine, and/or T may be replaced with U. In someembodiments region B comprises a trinucleotide of sequence AAA, AAT,AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG,TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT,TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG,CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT,GCC, GCG, GGA, GGT, GGC, and GGG wherein C may be 5-mthylcytosine and/orT may be replaced with U. In some embodiments region B comprises atrinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX,ATGX, ACAX, ACTX, ACCX, ACGX, AG AX, AGTX, AGCX, AGGX, TAAX, TATX, TACX,TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX,TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX,CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX,GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX, wherein X maybe selected from the group consisting of A, T, U, G, C and analoguesthereof, wherein C may be 5-mthylcytosine and/or T may be replaced withU. It will be recognized that when referring to (naturally occurring)nucleobases A, T, U, G, C, these may be substituted with nucleobaseanalogues which function as the equivalent natural nucleobase (e.g. basepair with the complementary nucleoside).

In some embodiments, the compound of the invention may comprise morethan one conjugate group (or more than one functional group X—such as aconjugate, targeting, blocking or activated group or a reactive oractivation group), such as 2 or 3 such groups. In some embodiments,region B is covalently linked, optionally via a [e.g. non-nucleotide]linker group), to at least one functional group, such as two or threefunctional groups. In some embodiments, the first region may becovalently linked (e.g. via internucleoside linkages, such asphosphodiester linkages), to two region Bs, for example, one 5′ and one3′ to the first region, wherein each region B may be (optionallyindependently) selected from the region B described herein. In thisrespect one region B may have one or more functional groups, and thesecond region B may have one or more function groups, wherein thefunctional groups of each region B may be independently selected from aconjugate, a targeting group, a blocking group or a reactive/activationgroup.

Poly Oligomeric Compounds

The invention provides for a poly oligomeric compound which may comprisethe first region (region A), the second region (region B) and the thirdregion (region C), wherein the first region is covalently linked to atleast one further oligomeric compound (region A′), wherein the firstregion (region A) and region A′ are covalently linked via a biocleavablelinker (region B′), which may be, by way of example, as according to thesecond region as disclosed here, for example a region of at least onephosphodiester linked DNA or RNA (such as DNA), such as two, three, fouror five phosphodiester linked DNA or RNA nucleosides (such as DNAnucleosides). Regions B and B′ may, in some embodiments have the samestructure, e.g. the same number of DNA/RNA nucleosides andphosphodiester linkages and/or the same nucleobase sequence. In otherembodiments Regions B and B′ may be different. By way of example suchpoly oligomeric compounds may have a structure such as: (5′-3′ or 3′-5′)Conjugate-PO—ON—PO′—ON′, wherein conjugate is region C, PO is region B,PO′ is region B′, and ON 1 is region A, and ON′ is region A′ It shouldbe understood that region A′ may, in some embodiments, comprise multiplefurther oligomeric compounds (such as a further 2 or 3 oligomericcompounds) linked in series (or in parallel) via biocleavable linkers,for example: Conjugate-PO—ON—PO—ON′—PO″—ON″, orConjugate-PO—ON—[PO—ON′]n, wherein n may, for example be 1, 2 or 3, andeach ON′ may be the same or different, and if different may have thesame or different targets.

Multi Conjugate Oligomeric Compounds

In some embodiments, the oligomeric compound may be conjugated to morethan one conjugate region (region C), which may be the same ordifferent. For example the oligomeric compound of the invention may havea structure as follows: (5′-3′ or 3′-5′) ON—PO′-Conj1-PO″-Conj2 whereinConj1 and conj2 are the two conjugate groups, at least one or both of POor PO″ are as according to region B herein, and ON is region A. Conj1and Conj2 may be the same or may be different. For example, in someembodiments, one of Conj1 and Conj2 are a carbohydrate or sterolconjugates and the other is a lipophilic conjugate, e.g. 5′-3′ or3′-5′:ON—PO′-Palmitoyl-PO″-Chol or ON—PO′-Palmitoyl-PO″-GalNac

The carbohydrate conjugate moiety (represented by GalNac in thepreceding formulas (e.g. when used as conj1 or conj2) may for example beselected from the group consisting of galactose, galactosamine,N-formyl-galactosamine, Nacetylgalactosamine, N-propionyl-galactosamine,N-n-butanoyl-galactosamine, and N-isobutanoylgalactose-amine. Thelipophilic conjugate (e.g. when used as conj1 or conj2, and representedas palmotoyl in the preceding formulas) may be a hydrophobic group, suchas a C16-20 hydrophobic group, a sterol, cholesterol. Other carbohydrateand lipophilic groups which may be used are, for example, disclosedherein.

The Target

In some embodiment, for a non-limiting example, the oligomer of theinvention is for use in modulating a nucleic acid (i.e. targets)selected from the group consisting of a mRNA, a microRNA, a IncRNA (longnon-coding RNA), a snRNA, snoRNA, and a viral RNA.

Exemplary, but not limiting mRNA and microRNA targets include forexample:

The genes indicated in cancer, such as Hif1-alpha, survivin, Bcl2, Mcl1,Her2, androgen receptor, beta-catenin, human transforming growth factorTGF-beta2, ras, TNF-alpha, c-RAF, HSPs e.g. Hsp27, elF-4E (e.g.ISIS-EIF4ER_(X)) STAT3 (e.g. ISIS-STAT3Rx), clusterin (e.g. OGX-011),AurkB, AurkA, PBK, miR-155, miR-21, miR-10b, mir-34 (see WO2011088309),miR-199a, miR-182,

The mRNAs of genes involved in inflammation, e.g. ICAM-1 (e.g.Alicoforsen), CD49d, VLA-4 osteopontin, miR-21 (psoriasis),

Other medically relevant mRNA targets include CTGF (local fibrosis) andc-Raf-kinase (ocular disease). miR-29 (cardiac fibrosis), Factor XI(clotting), factor VII (clotting) miR15 miR-159 (post-MI modeling(post-MI modeling), miR-138 (bone-loss), mir-21 (see WO12148952) andmir214 (fibrosis)—see WO2012012716.

Metabolic disease or disorders targets, such as Apo-B (high LDLcholesterol, ACS), ApoCIII (high serum TG, diabetes), Apo(a)(cardiovascular disease), FGFR4 (obesity), GCCR (T2 diabetes), GCGR (T2diabetes), PTP1B (T2 diabetes), DGAT2 (NASH), PCSK9 (hyperlipidaemia andrelated disorders), MtGPAT (obesity and NAFLD), miR-122 (highcholesterol), miR-33 (metabolic syndrome, atherosclerosis), miR-208(chronic heart failure), miR-499 (chronic heart failure), miR-378(cardio metabolic disease), mir-143 (vascular disease), miR-145(vascular disease), miR-92 (peripheral arterial disease), miR-375(diabetes), miR-27b (diabetes), miR-34a (diabetes), miR-199a, miR-27a(heart disease, ischemia), miR-338 (diabetes).

Metabolic diseases include, for examples, metabolic syndrome, obesity,hyperlipidemia, HDL/LDL cholesterol imbalance, dyslipidemias, e.g.,familial combined hyperlipidemia (FCHL), acquired hyperlipidemia,statin-resistant hypercholesterolemia, coronary artery disease (CAD),and coronary heart disease (CHD)., atherosclerosis, heart disease,diabetes (I and/or II), NASH, acute coronary syndrome (ACS), Viraldiseases: miR-451 (polycythemia), miR-122 (HCV), HBV, HCV, BKV, etc.Severe and rare diseases include SMN2 (spinal muscular atrophy), TTR(TTR amyloidosis), GHr (acromegaly), AAT (AATD associated liverdisease), Dystophin (Duchennes muscular dystrophy).

In some embodiments, the oligomer of the invention targets a liverexpressed nucleic acid, such as a liver expressed mRNA, such as PCSK9,ApoB, or MtGPAT. In some embodiments, the oligomer of the inventiontargets PCSK9 mRNA. In some embodiments, the oligomer of the inventiontargets ApoB mRNA. In some embodiments, the oligomer of the inventiontargets a liver expressed microRNA, such as miR-122.

In some embodiments, the oligomer of the invention is capable ofdown-regulating (e.g. reducing or removing) expression of the target(e.g. target nucleic acid). In this regards, the oligomer of theinvention can affect the inhibition of the target. In some embodiments,the oligomers of the invention bind to the target nucleic acid andaffect inhibition of expression of at least 10% or 20% compared to thenormal expression level, more preferably at least a 30%, 40%, 50%, 60%,70%, 80%, 90% or 95% inhibition compared to the normal expression level(such as the expression level in the absence of the oligomer(s) orconjugate(s)). In some embodiments, such modulation is seen when usingfrom 0.04 and 25 nM, such as from 0.8 and 20 nM concentration of thecompound of the invention. In the same or a different embodiment, theinhibition of expression is less than 100%, such as less than 98%inhibition, less than 95% inhibition, less than 90% inhibition, lessthan 80% inhibition, such as less than 70% inhibition. Modulation ofexpression level may be determined by measuring protein levels, e.g. bythe methods such as SDS-PAGE followed by western blotting using suitableantibodies raised against the target protein. Alternatively, modulationof expression levels can be determined by measuring levels of mRNA, e.g.by northern blotting or quantitative RT-PCR. When measuring via mRNAlevels, the level of down-regulation when using an appropriate dosage,such as from 0.04 and 25 nM, such as from 0.8 and 20 nM concentration,is, In some embodiments, typically to a level of from 10-20% the normallevels in the absence of the compound, conjugate or composition of theinvention.

The invention therefore provides a method of down-regulating orinhibiting the expression of the target in a cell which is expressingthe target, said method comprising administering the oligomer orconjugate according to the invention to said cell to down-regulating orinhibiting the expression of the target in said cell. Suitably the cellis a mammalian cell such as a human cell. The administration may occur,in some embodiments, in vitro. The administration may occur, in someembodiments, in vivo.

Compounds of the invention, such as the oligomers and conjugatesthereof, may be targeted to different targets, such as mRNA or microRNAor other nucleic acid targets which are expressed in the liver(references to NCBI Genbank/Gene IDs are given as examples of sequenceswhich may be targeted by the compounds of the invention—the Genbank/NCBIsequences are hereby incorporated by reference).

ApoB

In some embodiments, the first region (or first and second region) formsa single contiguous nucleobase sequence which is complementary, to acorresponding region of an ApoB mRNA target (i.e. targets) ApoB-100(NCBI Genbank ID NM_000384.2 GI: 105990531, hereby incorporated byreference).

Compounds of the invention which target ApoB may be used in thetreatment of acute coronary syndrome (see WO20100076248). The inventiontherefore provides for the oligomer according to the invention whichtargets ApoB100 for use in the treatment of acute coronary syndrome. Theinvention further provides for a method of treatment of acute coronarysyndrome, wherein said method comprises the administration of theoligomer of the invention to a subject in need to said treatment.

Compounds of the invention which target ApoB may be used in thetreatment atherosclerosis. The invention therefore provides for theoligomer according to the invention which targets ApoB100 for use in thetreatment of atherosclerosis. The invention further provides for amethod of treatment of atherosclerosis, wherein said method comprisesthe administration of the oligomer of the invention to a subject in needto said treatment.

Compounds of the invention which target ApoB may be used in thetreatment hypercholesterolemia or hyperlipidaemia. The inventiontherefore provides for the oligomer according to the invention whichtargets ApoB100 for use in the treatment of hypercholesterolemia orhyperlipidaemia. The invention further provides for a method oftreatment of hypercholesterolemia or hyperlipidaemia, wherein saidmethod comprises the administration of the oligomer of the invention toa subject in need to said treatment.

The invention provides for an in vivo or in vitro method for theinhibition of ApoB in a cell which is expressing ApoB, said methodcomprising administering an oligomer or conjugate or pharmaceuticalcomposition according to the invention to said cell so as to inhibitApoB in said cell.

Examples of LNA oligomers which may be used as the first region in theoligomers/conjugates of the invention include, for example thosedisclosed in WO2007/031081, WO2008/113830, WO2007131238, andWO2010142805, which are hereby incorporated by reference. Specificpreferred compounds include the following:

5′-G_(s) ^(m)C_(s)a_(s)t_(s)t_(s)g_(s)g_(s)t_(s)a_(s)t_(s)T_(s)^(m)C_(s)A-3′  (SEQ ID NO 1)

5-G_(s)T_(s)t_(s)g_(s)a_(s)C_(s)a_(s)C_(s)t_(s)g_(s)T_(s) ^(m)C-3  (SEQID NO 53)

Wherein capital letters are beta-D-oxy LNA units (nucleosides), lowercase letters are DNA units, subscript s is a phosphorothioate linkage,and a superscript m before the capital C illustrates that all LNAcytosines are 5-methyl cytosine. Compounds of the invention targetingApoB may be conjugated to a conjugate which targets the oligomer to theliver, as disclosed herein, such as a carbohydrate or lipophilicconjugate, such as a GalNac conjugate or a sterol conjugate (e.g.cholesterol or tocopherol). The conjugate may be, for example, at the 5′end or the 3′ end of the oligomer compound (suitably via region B).Other oligomers which target ApoB are disclosed in WO03/011887,WO04/044181, WO2006/020676, WO2007/131238, WO2007/031081, andWO2010142805.

PCSK9

In some embodiments, the first region (or first and second region) formsa single contiguous nucleobase sequence which is complementary, to acorresponding region of a PCSK9 mRNA target (i.e. targets), such as thehuman PCSK9 mRNA: NCBI Genbank ID NM_174936.3 GL299523249, herebyincorporated by reference.

The invention provides for an oligomer according to the invention whichtargets PCSK9, for use as a medicament, such as for the treatment ofhypercholesterolemia or related disorder, such as a disorder selectedfrom the group consisting of atherosclerosis, hyperlipidaemia,hypercholesterolemia, familiar hypercholesterolemia e.g. gain offunction mutations in PCSK9, HDL/LDL cholesterol imbalance,dyslipidemias, e.g., familial hyperlipidaemia (FCHL), acquiredhyperlipidaemia, statin-resistant hypercholesterolemia, coronary arterydisease (CAD), and coronary heart disease (CHD).

The invention provides for the use of an oligomer of the invention whichtargets PCSK9, for the manufacture of a medicament for the treatment ofhypercholesterolemia or a related disorder, such as a disorder selectedfrom the group consisting of atherosclerosis, hyperlipidaemia,hypercholesterolemia, familiar hypercholesterolemia e.g. gain offunction mutations in PCSK9, HDL/LDL cholesterol imbalance,dyslipidemias, e.g., familial hyperlipidaemia (FCHL), acquiredhyperlipidaemia, statin-resistant hypercholesterolemia, coronary arterydisease (CAD), and coronary heart disease (CHD).

The invention provides for a method of treating hypercholesterolemia ora related disorder, such as a disorder selected from the groupconsisting atherosclerosis, hyperlipidaemia, hypercholesterolemia,familiar hypercholesterolemia e.g. gain of function mutations in PCSK9,HDL/LDL cholesterol imbalance, dyslipidemias, e.g., familialhyperlipidaemia (FCHL), acquired hyperlipidaemia, statin-resistanthypercholesterolemia, coronary artery disease (CAD), and coronary heartdisease (CHD), said method comprising administering an effective amountof an oligomer according to the invention which targets PCSK9, to apatient suffering from, or likely to suffer from hypercholesterolemia ora related disorder.

The invention provides for an in vivo or in vitro method for theinhibition of PCSK9 in a cell which is expressing PCSK9, said methodcomprising administering an oligomer according to the invention whichtargets PCSK9 to said cell so as to inhibit PCSK9 in said cell.

The following is an oligomer which targets the human PCSK9 mRNA, and maybe used as region A in the compounds of the invention.

(SEQ ID NO 37) 5′-T_(s)G_(s)^(m)C_(s)t_(s)a_(s)c_(s)a_(s)a_(s)a_(s)a_(s)c_(s) ^(m)C_(s)^(m)C_(s)A-3′

Wherein capital letters are beta-D-oxy LNA units (nucleosides), lowercase letters are DNA units, subscript s is a phosphorothioate linkage,and a superscript m before the capital C illustrates that all LNAcytosines are 5-methyl cytosine. Compounds of the invention targetingPCSK9 may be conjugated to a conjugate which targets the oligomer to theliver, as disclosed herein, such as a carbohydrate or lipophilicconjugate, such as a GalNac conjugate or a sterol conjugate (e.g.cholesterol or tocopherol). The conjugate may be, for example, at the 5′end or the 3′ end of the oligomer compound (suitably via region B).Other oligomers which target PCSK9 are disclosed as the SEQ ID NO 36-52,and others are disclosed in WO2008/043753, WO2011/009697, WO08/066776,WO07/090071, WO07/146511, WO07/143315, WO09/148605, WO11/123621, andWO11133871, which are hereby incorporated by reference.

miR-122

In some embodiments, the first region (or first and second region) forma single contiguous nucleobase sequence which is complementary, to acorresponding region of a microRNA-122 such as miR-122a (i.e. targets),such as the has-miR-122 sequences (miRBase release 20: MI0000442), suchas:

>hsa-mir-122 MI0000442CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC >hsa-miR-122-5p MIMAT0000421UGGAGUGUGACAAUGGUGUUUG

miR-122 has been indicated in HCV infection, where it is an essentialhost factor required for maintenance of the infection. Inhibitors ofmiR-122 may therefore be used in the treatment of hepatitis C infection.

Compounds of the invention which target miR-122 may be used in thetreatment of HCV infection. The invention therefore provides for theoligomer according to the invention which targets miR-122 for use in thetreatment of HCV infection. The invention further provides for a methodof treatment of HCV infection, wherein said method comprises theadministration of the oligomer of the invention to a subject in need tosaid treatment.

The invention provides for the use of an oligomer of the invention whichtargets miR-122, for the manufacture of a medicament for the treatmentof HCV infection.

The invention provides for a method of treating HCV infection, saidmethod comprising administering an effective amount of an oligomeraccording to the invention which targets miR-122, to a patient sufferingfrom HCV infection.

The invention provides for an in vivo or in vitro method for theinhibition of miR-122 in a cell which is expressing miR-122, such as anHCV infected cell or a HCV replicon expressing cell, said methodcomprising administering an oligomer or conjugate or pharmaceuticalcomposition according to the invention to said cell so as to inhibitmiR-122 in said cell.

miR-122 has also been indicated in cholesterol metabolism, and it hasbeen suggested that inhibition of miR-122 may be used for a treatment toreduce plasma cholesterol levels (Esau, Cell Metab. 2006 February;3(2):87-98.)

Inhibitors of miR-122 may therefore be used in a treatment to reduceplasma cholesterol levels, or in the treatment of a metabolic diseaseassociated with elevated levels of cholesterol (related disorders), suchas indications selected from the group consisting of atherosclerosis,hyperlipidaemia, hypercholesterolemia, familiar hypercholesterolemia,dyslipidemias, coronary artery disease (CAD), and coronary heart disease(CHD) Compounds of the invention which target miR-122 may be used in thetreatment of elevated cholesterol levels or related disorders. Theinvention therefore provides for the oligomer according to the inventionwhich targets miR-122 for use in the treatment of elevated cholesterollevels or related disorders. The invention further provides for a methodof treatment of elevated cholesterol levels or related disorders,wherein said method comprises the administration of the oligomer of theinvention to a subject in need to said treatment.

The invention provides for the use of an oligomer of the invention whichtargets miR-122, for the manufacture of a medicament for the treatmentof elevated cholesterol levels or related disorders.

The invention provides for a method of treating elevated cholesterollevels or related disorders, said method comprising administering aneffective amount of an oligomer according to the invention which targetsmiR-122, to a patient suffering from said disorder.

The invention provides for an in vivo or in vitro method for theinhibition of miR-122 in a cell which is expressing miR-122, such as anHCV infected cell or a HCV replicon expressing cell, said methodcomprising administering an oligomer or conjugate or pharmaceuticalcomposition according to the invention to said cell so as to inhibitmiR-122 in said cell.

Oligomer's targeting miR-122 are disclosed in WO2007/112754,WO2007/112753, WO2009/043353, and may be mixmers, such as SPC3649, alsoreferred to as miravirsen see below, or a tiny LNA, such as thosedisclosed in WO2009/043353 (e.g. 5′-ACACTCC-3′, 5′-CACACTCC-3′,5′-TCACACTCC-3′, where capital letters are beta-D_oxy LNA, fullyphosphorothioate and LNA C are 5-methyl cytosine). In some embodiments,the miR-122 targeting oligomers have a length of 8, 9, 10, 11, 12, 13,14, 15, 16, 17 or 18 (or 19, 20, 21, 22 or 23 nucleotides) in length. Insome embodiments, the miR-122 targeting oligomers a sequence which isfully complementary to miR-122 as measured across the length of theoligomer, and preferably include the sequence 5′-CACACTCC-3′. In someembodiments, the oligomer targeting a microRNA such as miR-122, iscomplementary to a corresponding region of the microRNA across thelength of the oligomer and in some embodiments the 3′ nucleoside of theoligomer is compelmentary to (i.e. aligns to) the first, second, thirdor fourth 5′ nucleotides of the microRNA, such as miR-122, such as thesecond 5′ nucleotide of the microRNA, such as miR-122.

The following is an oligomers which targets the has-miR-122 (humanmiR-122), and may be used as region A in the compounds of the invention.

Miravirsen: 5′-^(m)C_(s)c_(s)A_(s)t_(s)t_(s)G_(s)T_(s)c_(s)a_(s)^(m)C_(s)a_(s) ^(m)C_(s)t_(s) ^(m)C_(s) ^(m)C-3′

Other miR-122 targeting compounds which may be used in the context ofthe present invention (region A) are disclosed in WO2007/027894,WO2007/027775.

MtGPAT: (NCBI gene ID 57678—Chromosome: 10;NC_000010.10(113907971..113975153, complement) Mitochondrialglycerol-3-phosphate acyltransferase 1 (EC 2.3.1.15, also known asGPAT1, mtGPAT1, GPAM, mtGPAM) plays a major role in hepatic triglycerideformation, where high levels of mtGPAT1 activity results in fatty liver(hepatosteatosis) whereas the absence of mtGPAT1 results in low levelsof liver triglycerides and stimulated fatty acid oxidation (seeWO2010/000656 which discloses oligomers which target mtGPAT. Compoundsof the invention which target MtGPAT may be used to treat conditionssuch as being overweight, obesity, fatty liver, hepatosteatosis, nonalcoholic fatty liver disease (NAFLD), non alcoholic steatohepatitis(NASH), insulin resistance, diabetes such as non insulin dependentdiabetes mellitus (NIDDM)

FactorVII (NCBI Gene ID 2155, NCBI J02933.1 GI: 180333, or EU557239.1GI: 182257998). The oligomer or conjugate of the invention may targetFactorVII, and thereby inhibit the production of FactorVII, a keycomponent of the tissue factor coagulation pathway. Compounds of theinvention which target FactorVII may be used for the treatment orprevention of thrombotic diseases (typically without causing bleeding)and as heart attack, stroke and blood clots, or inflammatory conditions.WO 2013/119979 and WO 2012/174154, hereby incorporated by referencedisclose oligonucleotide compounds which target FVII which may beincorporated into the conjugates of the present invention.

Factor XI (NCBI Genbank BC122863.1 GI: 114108211)—Factor XI, a clottingfactor that is produced in the liver. High levels of Factor XI arelinked to heart attack, stroke and blood clots. WO 2013/070771, herebyincorporated by reference, discloses oligonucleotide compounds whichtarget XI which may be incorporated into the conjugates of the presentinvention. Compounds of the invention which target Factor XI may be usedfor the treatment or prevention of thrombotic diseases, and as heartattack, stroke and blood clots, or inflammatory conditions such asarthritis and colitis.

ApoCIII (NCBI Genbank BC027977.1 GI: 20379764) a protein that regulatestriglyceride metabolism in blood. High levels of apoC-III are linked toinflammation, high triglycerides, atherosclerosis and metabolicsyndrome. Compounds of the invention which target ApoCIII may be used toreduce serum triglyceride levels or in the treatment of e.g. familialchylomicronemia syndrome and severely high triglycerides either as asingle agent or in combination with other triglyceride-lowering agents.WO11085271 hereby incorporated by reference, discloses oligonucleotidecompounds which target ApoCIII which may be incorporated into theconjugates of the present invention.

Apo(a) (NCBI Genbank NM_005577.2 GI: 116292749) inhibits the productionof apo(a) in the liver and is designed to offer a direct approach toreducing Lp(a), an independent risk factor for cardiovascular disease.High levels of Lp(a) are associated with an increased risk ofatherosclerosis, coronary heart disease, heart attack and stroke. Lp(a)promotes premature plaque buildup, or atherosclerosis, in arteries.Compounds of the invention which target Apo(a) may be used in thetreatment of e.g. atherosclerosis and coronary heart disease. WO05000201and WO03014307 hereby incorporated by reference, disclosesoligonucleotide compounds which target apolipoprotein (a) which may beincorporated into the conjugates of the present invention.

Hepatitis B (HBV) (see for example NCBI D23684.1 GI: 560092; D23683.1GI: 560087; D23682.1 GI: 560082; D23681.1 GI: 560077; D23680.1 GI:560072; D23679.1 GI: 560067; D23678.1 GI: 560062; D23677.1 GI: 560057;all of which are hereby incorporated by reference)

Oligomers which target HBV are well known in the art, for example see,WO96/03152, WO97/03211, WO2011/052911, WO2012/145674, WO2012/145697,WO2013/003520 and WO2013/159109.

Compounds of the invention which target HBV may be used in the treatmentHBV infection. The invention therefore provides for the oligomeraccording to the invention which targets HBV for use in the treatment ofHBV. The invention further provides for a method of treatment of HBVinfection, wherein said method comprises the administration of theoligomer of the invention to a subject in need to said treatment.

The invention provides for the oligomer or conjugate of the inventionwhich targets hepatitis B (HBV) for use as a medicament, such as for thetreatment hepatitis B infection or a related disorder.

The invention provides for the use of an oligomer or conjugate orpharmaceutical composition according to the invention which targetshepatitis B (HBV), for the manufacture of a medicament for the treatmentof hepatitis B infection or a related disorder.

The invention provides for a method of treating treatment hepatitis Binfection or a related disorder, said method comprising administering aneffective amount of an oligomer or conjugate of the invention whichtargets HBV, to a patient infected with Hepatitis B virus.

The invention provides for an in vivo or in vitro method for theinhibition of HBV replication in a cell infected with HBV, said methodcomprising administering an oligomer or conjugate of the invention whichtargets HBV to said cell so as to inhibit HBV replication. An example ofan LNA oligomer which target's HBV is (as is disclosed in WO2011/47312)which may be used as the oligomer (region A) of the invention5′-G_(s)A_(s)G_(s)G_(s)C_(s)a_(s)t_(s)a_(s)g_(s)C_(s)a_(s)g_(s)^(m)C_(s)A_(s)G_(s)G-3′. Further compounds are disclosed in table 1 ofWO2011/47312, and in WO2011/052911, WO2012/145674, WO2012/145697,WO2013/003520 and WO2013/159109, hereby incorporated by reference.

RG-101 is a compound which targets miR-122 and comprises a GalNacconjugate, and is being developed for treatment of HCV by RegulusTherapeutics.

ANGPTL3, (e.g. NCBI BC007059.1 GI: 14712025 or BC058287.1 GI: 34849466)ANGIOPOIETIN-UKE 3—a protein that regulates lipid, glucose and energymetabolism. Humans with elevated levels of ANGPTL3 have hyperlipidemiaassociated with an increased risk of premature heart attacks, increasedarterial wall thickness as well as multiple metabolic abnormalities,such as insulin resistance. In contrast, humans with lower levels ofANGPTL3 have lower LDL-C and triglyceride levels and a lower risk ofcardiovascular disease. Compounds of the invention which target ANGPTL3may be used in the treatment of e.g. hyperlipidemia and relateddisorders, metabolic disorder, atherosclerosis, coronary heart diseaseor insulin resistance. WO11085271 hereby incorporated by reference,discloses oligonucleotide compounds which target ANGPTL3 which may beincorporated into the conjugates of the present invention.

Glucagon receptor, or GCGR (BC112041.1 GI: 85567507; L20316.1 GI:405189): Glucagon is a hormone that opposes the action of insulin andstimulates the liver to produce glucose, particularly in type 2diabetes. In patients with advanced diabetes, uncontrolled glucagonaction leads to a significant increase in blood glucose levels.Therefore, attenuating glucagon action may have a significant glucoselowering effect in patients with severe diabetes. In addition, reducingGCGR produces more active glucagon-like peptide, or GLP-1, a hormonethat preserves pancreatic function and enhances insulin secretion.Compounds of the invention which target GCGR may be used in thetreatment of e.g. or insulin resistance, hyperglycemia, diabetes, suchas type 1 or 2 diabetes, preservation of pancreatic function, and tocontrol of blood glucose levels. WO2007/134014 discloses oligonucleotidecompounds which target GCGR which may be incorporated into theconjugates of the present invention.

Fibroblast growth factor receptor 4, or FGFR4. (NCBI Gene2264—NC_000005.9 (176513906..176525143) FGFR4 is expressed in the liverand fat tissues, and is indicated in decreasing the body's ability tostore fat while simultaneously increasing fat burning and energyexpenditure. Many anti-obesity drugs act in the brain to suppressappetite, commonly resulting in CNS side effects. Compounds of theinvention which target FGFR4 may be used in the treatment of e.g. orinsulin resistance, hyperglycemia, diabetes, such as type 1 or 2diabetes, preservation of obesity (e.g. when used in combination with anappetite-suppressing drug), reducing body weight, and improvement ininsulin sensitivity, diabetes, such as type 1 or 2 diabetes and tocontrol of blood glucose levels. WO09046141 and WO12174476 herebyincorporated by reference disclose oligonucleotide compounds whichtarget FGFR4 which may be incorporated into the conjugates of thepresent invention.

Diacylglycerol acyltransferase-2, or DGAT-2 (NCBI GENE ID 84649): A keycomponent in the synthesis of triglycerides. The inhibition of DGAT mayreduce liver fat in patients with Nonalcoholic Steatohepatitis (NASH),and may also be used to treat type 2 diabetes and insulin resistance.Compounds of the invention which target DGAT-2 may be used to treatNASH, to reduce liver fat, to treat diabetes, such as type 2 diabetes,and treat insulin resistance. WO05019418 and WO2007136989, herebyincorporated by reference disclose oligonucleotide compounds whichtarget DGAT-2 which may be incorporated into the conjugates of thepresent invention.

Glucocorticoid receptor, or GCCR (BC150257.1 GI: 152013043):Glucocorticoid hormones affect a variety of processes throughout thebody, and excessive levels of glucocorticoid hormones can have adetrimental effect on many of the tissues and organs in the body.Cushing's Syndrome is an orphan disease caused by prolonged exposure tohigh levels of glucocorticoids. If untreated, patients with Cushing'sSyndrome can develop hypertension, diabetes and impaired immunefunctions and have an increased risk of early death. Although there areapproved treatments for Cushing's Syndrome, current medicines areassociated with significant side effects, such as hypertension anddiabetes, and there remains a high unmet medical need for new therapiesfor these patients. Compounds of the invention which target GCCR-2 maybe used to treat Cushing's Syndrome and associated conditions (such asthose listed above). WO07035759 and WO2007136388, which are herebyincorporated by reference disclose oligonucleotide compounds whichtarget GCCR which may be incorporated into the conjugates of the presentinvention.

Complement component C5 (M57729.1 GI: 179982): The complement systemplays a central role in immunity as a protective mechanism for hostdefense, but its dysregulation results in serious, life-threateningcomplications in a broad range of human diseases including paroxysmalnocturnal hemoglobinuria (PNH), atypical hemolytic-uremic syndrome(aHUS), myasthenia gravis, neuromyelitis optica, amongst others.Compounds of the invention which target complement component C5 may beused to treat one or more of these disorders. C5 is a genetically andclinically validated target; loss of function human mutations areassociated with an attenuated immune defense against certain infectionsand intravenously administered anti-C5 monoclonal antibody therapy hasdemonstrated clinical activity and tolerability in a number ofcomplement-mediated diseases, transmembrane protease, serine 6 (Tmprss6)for the treatment of beta-thalassemia and iron-overload disorders.

Alpha-1 antitrypsin (AAT): (M11465.1 GI: 177826) Liver diseaseassociated with—WO13142514 which is hereby incorporated by referencedisclose oligonucleotide compounds which target AAT which may beincorporated into the oligomers or conjugates of the present invention.Compounds of the invention which target AAT may be used in methods fordecreasing AIAT mRNA and protein expression and treating, ameliorating,preventing, slowing progression, or stopping progression of fibrosis,such as, AIATD associated liver disease, and pulmonary disease, such as,AIATD associated pulmonary disease in an individual in need thereof.

Transthyretin—TTR (BC005310.1 GI: 13529049): The oligomers of theinvention which target TTR may be used to treat transthyretinamyloidosis, or TTR amyloidosis, a severe and rare genetic disease inwhich the patient inherits a mutant gene that produces a misfolded formof TTR, which progressively accumulates in tissues. In patients with TTRamyloidosis, both the mutant and normal forms of TTR can build up asfibrils in tissues, including heart, peripheral nerves, and thegastrointestinal tract. The presence of TTR fibrils interferes with thenormal functions of these tissues, and as the TTR protein fibrilsenlarge more tissue damage occurs and the disease worsens. TTR is acarrier protein that transports a thyroid hormone and retinol in theblood. In patients with TTR amyloidosis, both the mutant and normalforms of TTR can build up as fibrils in tissue. The compounds of theinvention may be used to treat TTR amyloidosis. See Benson et al.,Amyloid. 2010 June; 17(2):43-9, and Ackermann et al., Amyloid. 2012June; 19 Suppl 1:43-4.). Antisense compounds targeting TTR which may beused in the oligomers or conjugates of the invention are disclosed inU.S. Pat. No. 8,101,743, WO11139917 and WO10017509, which are herebyincorporated by reference.

Ammolevulinate synthase-1 (ALAS-1) (BC011798.2 GI: 33877783; AK312566.1GI: 164690365; NM_199166.2 GI: 362999012; NM_000688.5 GI: 362999011).ALAS1 is a validated target for the treatment of porphyria, such as thetreatment of hepatic porphyrias including acute intermittent porphyria(AIP). Compounds of the invention which target ALAS-1 may be used in thetreatment of these disorders.

Vascular endothelial growth factor, or VEGF (GENE ID 7422, humanSequence: Chromosome: 6; NC_000006.11 (43737946..43754224)). VEGF isindicated in cancers. Compounds of the invention which target VEGF maybe used in the treatment of hyperproliferative disorders, such ascancer, such as liver cancer.

Table 1 provides for a group of liver targets which may be targeted bythe compounds of the invention, as well as the medicalindication/disorder for which such compounds may be used to treat (suchas a person suffering from the associated disorder) (See Sehgal et al.,Liver as a target for oligonucleotide therapeutics, J. of Hepatology2013, In Press).

TABLE 1 The compound of the invention may target a nucleic acid (e.g.mRNA encoding, or miRNA) For the treatment of a disease or selected fromthe group consisting of disorder such as AAT AAT-LivD ALDH2 Alcoholdependence HAMP pathway Anemia or inflammation/CKD miR-33Atherosclerosis Apo(a) Atherosclerosis/high Lp(a) miR-7 Liver cancermiR-378 Cardiometabolic diseases miR-21 Liver cancer Myc Liver cancermiR-122 HCV 5′UTR HCV 5′UTR & NS5B HCV NS3 HCV TMPRSS6 HemochromatosisAntithrombin III Hemophilia A, B ApoCIII Hypertriglyceridemia ANGPLT3Hyperlipidemia MTP Hyperlipidemia DGAT2 NASH ALAS1 PorphyriaAntithrombin III Rare Bleeding disorders Serum amyloid A SAA-amyloidosisFactor VII Thrombosis Growth hormone receptor Acromegaly miR-122Hepatitis C virus ApoB-100 Hypercholesterolemia ApoCIIIHypertriglyceridemia PCSK9 Hypercholesterolemia CRP Inflammatorydisorders KSP or VEGF Liver cancer PLK1 Liver cancer miR-34 Liver cancerFGFR4 Obesity Factor IXa Thrombosis Factor XI Thrombosis TTR TTRamyloidosis GCCR Type 2 diabetes PTP-1B Type 2 diabetes GCGR Cushing'sSyndrome Hepatic Glucose 6-Phosphate glucose homeostasis, diabetes, typeTransporter-1 2 diabetes

Sequences

In some embodiments, the oligomers, or first region thereof, comprise acontiguous nucleotide sequence which corresponds to the reversecomplement of a nucleotide sequence present in the target nucleic acid(i.e. the sequence which the oligomer targets). Table 3 provides a groupof mRNA and miRNA targets which are in pre-clinical or clinicaldevelopment using oligonucleotide compounds for the associatedindication, and are therefore suitable for targeting with the compoundsof the present invention.

In some embodiments the target is selected from the group consisting of:miR-122, ApoB-100, ApoCIII, PCSK9, CRP, KSP, VEGF, PLK1, miR-34, FGFR4,Factor IXa, Factor XI, TTR, GCCR, PTP-1B, GCGR, AAT, ALDH2, HAMPpathway, miR-33, Apo(a), miR-7, miR-378, miR-21, Myc, miR-122, the HCVgenome such as the HCV 5′UTR or HCV NS5B RNA or NS3 RNA, TMPRSS6,Antithrombin III, ApoCIII, ANGPLT3, MTP, DGAT2, ALAS1, Antithrombin III,Serum amyloid A and Factor VII.

In some embodiments, the contiguous nucleotide sequence comprises nomore than a single mismatch when hybridizing to the target sequence.Region B may however be non-complementary and may therefore bedisregarded when determining the degree of complementarity.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target region of the nucleicacid, such as those disclosed herein, the degree of “complementarity”(also, “homology” or “identity”) is expressed as the percentage identity(or percentage homology) between the sequence of the oligomer (or regionthereof) and the sequence of the target region (or the reversecomplement of the target region) that best aligns therewith. Thepercentage is calculated by counting the number of aligned bases thatare identical between the 2 sequences, dividing by the total number ofcontiguous monomers in the oligomer, and multiplying by 100. In such acomparison, if gaps exist, it is preferable that such gaps are merelymismatches rather than areas where the number of monomers within the gapdiffers between the oligomer of the invention and the target region.

As used herein, the terms “homologous” and “homology” areinterchangeable with the terms “identity” and “identical”.

The terms “corresponding to” and “corresponds to” refer to thecomparison between the nucleotide sequence of the oligomer (i.e. thenucleobase or base sequence) or contiguous nucleotide sequence (a firstregion) and the equivalent contiguous nucleotide sequence of a furthersequence selected from either i) a sub-sequence of the reversecomplement of the nucleic acid target. Nucleotide analogues are compareddirectly to their equivalent or corresponding nucleotides. A firstsequence which corresponds to a further sequence under i) or ii)typically is identical to that sequence over the length of the firstsequence (such as the contiguous nucleotide sequence) or, as describedherein may, in some embodiments, is at least 80% homologous to acorresponding sequence, such as at least 85%, at least 90%, at least91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%homologous, such as 100% homologous (identical).

The terms “corresponding nucleotide analogue” and “correspondingnucleotide” are intended to indicate that the nucleotide in thenucleotide analogue and the naturally occurring nucleotide areidentical. For example, when the 2-deoxyribose unit of the nucleotide islinked to an adenine, the “corresponding nucleotide analogue” contains apentose unit (different from 2-deoxyribose) linked to an adenine.

The terms “reverse complement”, “reverse complementary” and “reversecomplementarity” as used herein are interchangeable with the terms“complement”, “complementary” and “complementarity”.

The contiguous nucleobase sequence of the oligomer (first region orfirst and second region) may therefore be complementary to a target,such as those referred to herein.

In some embodiments, the first region or first and second region form asingle contiguous nucleobase sequence which is complementary to a regionof a mRNA target, such as those referred to herein, including, forexample, ApoB-100 (NM_000384.2 GI: 105990531 or PCSK9 (NM_174936.3 GI:299523249).

Length

The oligomers may comprise or consist of a contiguous nucleotidesequence of a total of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 contiguousnucleotides in length.

In some embodiments, the oligomers comprise or consist of a contiguousnucleotide sequence of a total of from 10-22, such as 12-18, such as13-17 or 12-16, such as 13, 14, 15, 16 contiguous nucleotides in length.

In some embodiments, the oligomers comprise or consist of a contiguousnucleotide sequence of a total of 10, 11, 12, 13, or 14 contiguousnucleotides in length.

In some embodiments, the oligomer according to the invention consists ofno more than 22 nucleotides, such as no more than 20 nucleotides, suchas no more than 18 nucleotides, such as 15, 16 or 17 nucleotides. Insome embodiments the oligomer of the invention comprises less than 20nucleotides. It should be understood that when a range is given for anoligomer, or contiguous nucleotide sequence length it includes the loweran upper lengths provided in the range, for example from (or between)10-30, includes both 10 and 30.

Nucleosides and Nucleoside Analogues

The term “nucleotide” as used herein, refers to a glycoside comprising asugar moiety (or analogue thereof), a base moiety and a covalentlylinked group (linkage group), such as a phosphate or phosphorothioateinternucleotide linkage group, and covers both naturally occurringnucleotides, such as DNA or RNA, and non-naturally occurring nucleotidescomprising modified sugar and/or base moieties, which are also referredto as “nucleotide analogues” herein. Herein, a single nucleotide (unit)may also be referred to as a monomer or nucleic acid unit.

It will be recognized that in the context of the present invention theterm nucleoside and nucleotide are used to refer to both naturallyoccurring nucleotides/sides, such as DNA and RNA, as well asnucleotide/side analogues.

In field of biochemistry, the term “nucleoside” is commonly used torefer to a glycoside comprising a sugar moiety and a base moiety, andmay therefore be used when referring to the nucleotide units, which arecovalently linked by the internucleoside linkages between thenucleotides of the oligomer. In the field of biotechnology, the term“nucleotide” is often used to refer to a nucleic acid monomer or unit,and as such in the context of an oligonucleotide may refer to thebase—such as the “nucleotide sequence”, typically refer to thenucleobase sequence (i.e. the presence of the sugar backbone andinternucleoside linkages are implicit). Likewise, particularly in thecase of oligonucleotides where one or more of the internucleosidelinkage groups are modified, the term “nucleotide” may refer to a“nucleoside” for example the term “nucleotide” may be used, even whenspecifying the presence or nature of the linkages between thenucleosides.

As one of ordinary skill in the art would recognize, the 5′ terminalnucleotide of an oligonucleotide does not comprise a 5′ internucleosidelinkage group, although may or may not comprise a 5′ terminal group.

Non-naturally occurring nucleotides include nucleotides which havemodified sugar moieties, such as bicyclic nucleotides or 2′ modifiednucleotides, such as 2′ substituted nucleotides.

“Nucleotide analogues” are variants of natural nucleotides, such as DNAor RNA nucleotides, by virtue of modifications in the sugar and/or basemoieties. Analogues could in principle be merely “silent” or“equivalent” to the natural nucleotides in the context of theoligonucleotide, i.e. have no functional effect on the way theoligonucleotide works to inhibit target gene expression. Such“equivalent” analogues may nevertheless be useful if, for example, theyare easier or cheaper to manufacture, or are more stable to storage ormanufacturing conditions, or represent a tag or label. Preferably,however, the analogues will have a functional effect on the way in whichthe oligomer works to inhibit expression; for example by producingincreased binding affinity to the target and/or increased resistance tointracellular nucleases and/or increased ease of transport into thecell. Specific examples of nucleoside analogues are described by e.g.Freier & Altmann; Nucl. Add Res., 1997, 25, 4429-4443 and Uhlmann; Curr.Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1:

The oligomer may thus comprise or consist of a simple sequence ofnatural occurring nucleotides—preferably 2′-deoxynucleotides (referredhere generally as “DNA”), but also possibly ribonucleotides (referredhere generally as “RNA”), or a combination of such naturally occurringnucleotides and one or more non-naturally occurring nucleotides, i.e.nucleotide analogues. Such nucleotide analogues may suitably enhance theaffinity of the oligomer for the target sequence.

Examples of suitable and preferred nucleotide analogues are provided byWO2007/031091 or are referenced therein. Other nucleotide analogueswhich may be used in the oligomer of the invention include tricyclicnucleic acids, for example please see WO2013154798 and WO2013154798which are hereby incorporated by reference.

Incorporation of affinity-enhancing nucleotide analogues in theoligomer, such as LNA or 2′-substituted sugars, can allow the size ofthe specifically binding oligomer to be reduced, and may also reduce theupper limit to the size of the oligomer before non-specific or aberrantbinding takes place.

Oligomeric compounds, such as antisense oligonucleotides, such as thecompounds referred to herein, including region A, and in some optionalembodiments, region B, may contain one or more nucleosides wherein thesugar group has been modified. Such sugar modified nucleosides(nucleoside analogues) may impart enhanced nuclease stability, increasedbinding affinity, or some other beneficial biological property to theantisense compounds. In some embodiments, nucleosides comprise achemically modified ribofiiranose ring moiety.

In some embodiments, the oligomer, or first region thereof, comprises atleast one, such as at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 9, at least 10, at least 11,at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24 or 25 nucleoside analogues, such as sugarmodified nucleoside analogues.

Bicyclic nucleoside analogues include nucleoside analogues whichcomprise a bridge (or biradical) linking the second and forth carbon ofthe ribose ring, (C4*-C2* bridge or biradical). The presence of thebiradical between the 2^(nd) and 4^(th) carbon locks the ribose into a3′ endo-(north) confirmation, and as such bicyclic nucleoside analogueswith a C2*-C4* biradical are often referred to as Locked nucleic acid(LNA). In some embodiments the nucleoside analogues are (optionallyindependently selected from the group consisting of bicyclic nucleosideanalogues (such as LNA), and/or 2′ substituted nucleoside analogues,such as (optionally independently) selected from the group consisting of2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, 2′-AP,2′-FANA, 2′-(3-hydroxy)propyl, and 2′-fluoro-DNA units, and/or other(optionally) sugar modified nucleoside analogues such as morpholino,peptide nucleic acid (PNA), CeNA, unlinked nucleic acid (UNA), hexitolnucleoic acid (HNA). bicyclo-HNA (see e.g. WO2009/100320), In someembodiments, the nucleoside analogues increase the affinity of the firstregion for its target nucleic acid (or a complementary DNA or RNAsequence).

In some embodiments, the oligomer comprises at least one bicyclicnucleotide analogue, such as LNA. In some embodiments, the first regioncomprises of at least one bicyclic nucleoside analogues (e.g. LNA)and/or 2′substituted nucleoside analogues. In some embodiments, thenucleoside analogues present in the oligomer all comprise the same sugarmodification. In some embodiments, at least one nucleoside analoguepresent in the first region is a bicyclic nucleoside analogue, such asat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 11, at least 12, at least13, at least 14, at least 15, at least 16, for example all nucleosideanalogues (except the DNA and or RNA nucleosides of region B) are sugarmodified nucleoside analogues, such as such as bicyclic nucleosideanalogues, such as LNA, e.g. beta-D-X-LNA or alpha-L-X-LNA (wherein X isoxy, amino orthio), or other LNAs disclosed herein including, but notlimited to, (R/S) cET, cMOE or 5′-Me-LNA.

Examples of chemically modified ribofiiranose rings include, withoutlimitation, addition of substituent groups (including 5′ and 2′substituent groups); bridging of non-geminal ring atoms to form bicyclicnucleic acids (BNA); replacement of the ribosyl ring oxygen atom with S,N(R), or C(R₁)(R₂) (R═H, C₁-C₂ alkyl or a protecting group); andcombinations thereof. Examples of chemically modified sugars include,2′-F-5′-methyl substituted nucleoside (see, PCT InternationalApplication WO 2008/101157, published on Aug. 21, 2008 for otherdisclosed 5′, 2′-bis substituted nucleosides), replacement of theribosyl ring oxygen atom with S with further substitution at the2′-position (see, published U.S. Patent Application US2005/0130923,published on Jun. 16, 2005), or, alternatively, 5′-substitution of a BNA(see, PCT International Application WO 2007/134181, published on Nov.22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a5′-vinyl group).

Examples of nucleosides having modified sugar moieties include, withoutlimitation, nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S,2′-F, 2′-OCH₃, and 2′-O(CH₂)2O CH₃ substituent groups. The substituentat the 2′ position can also be selected from allyl, amino, azido, thio,O-allyl, O—C₁-C₁₀ alkyl, OCF₃, O(CH₂)₂SCH₃, O(CH₂)2-O—N(Rm)(Rn), andO—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H orsubstituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosidescomprising a bicyclic sugar moiety. Examples of bicyclic nucleosidesinclude, without limitation, nucleosides comprising a bridge between the4′ and the 2′ ribosyl ring atoms. In some embodiments, compoundsprovided herein include one or more bicyclic nucleosides wherein thebridge comprises a 4′ to 2′ bicyclic nucleoside. Examples of such 4′ to2′ bicyclic nucleosides, include, but are not limited to, one of theformulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA);4′-CH(CH₃)—O-2′ and 4′-CH(CH₂OCH₃)—O-2*, and analogs thereof (see, U.S.Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′, andanalogs thereof (see, published PCT International ApplicationWO2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′, and analogsthereof (see, published PCT International Application WO2008/150729,published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see, published U.S. PatentApplication US2004/0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₀ alkyl, or a protecting group (see, U.S. Pat. No.7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see,Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′, and analogs thereof (see, published PCT InternationalApplication WO 2008/154401, published on Dec. 8, 2008). Also see, forexample: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett.,1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Srivastava et al., J. Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4, 2007);Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braaschet al., Chem. Biol, 2001, 8, 1-7; Oram et al, Curr. Opinion Mol. Ther.,2001, 3, 239-243; U.S. Pat. Nos. 6,670,461, 7,053,207, 6,268,490,6,770,748, 6,794,499, 7,034,133, 6,525,191, 7,399,845; published PCTInternational applications WO 2004/106356, WO 94/14226, WO 2005/021570,and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570,US2007/0287831, and US2008/0039618; and U.S. patent Ser. Nos.12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564,61/086,231,61/097,787, and 61/099,844; and PCT International ApplicationNos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922. Eachof the foregoing bicyclic nucleosides can be prepared having one or morestereochemical sugar configurations including for exampleα-L-ribofuranose and beta-D-ribofuranose (see PCT internationalapplication PCT DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In some embodiments, bicyclic sugar moieties of BNA nucleosides include,but are not limited to, compounds having at least one bridge between the4′ and the 2′ position of the pentofuranosyl sugar moiety wherein suchbridges independently comprises 1 or from 2 to 4 linked groupsindependently selected from —[CiR_(a)XR_(b))],,—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)x-, and —N(Ra)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4;each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-Ci₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-Ci₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and each J₁ and J₂ is, independently, H, C₁-C₆alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂o aryl, acyl (C(═O)—H), substituted acyl, a heterocycleradical, a substituted heterocycle radical, C1-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkyl, or a protecting group.

In some embodiments, the bridge of a bicyclic sugar moiety is,—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—. In some embodiments,the bridge is 4′-CH₂-2′, 4′-(CH₂)2-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′,4*-(CH₂)2-O-2′, 4′-CH₂—O—N(R)- 2′, and 4′-CH₂—N(R)—O-2′-, wherein each Ris, independently, H, a protecting group, or C₁-C₁₂ alkyl.

In some embodiments, bicyclic nucleosides are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the a-L configuration or in the beta-Dconfiguration. Previously, a-L-methyleneoxy (4′-CH₂-0-2′) BNA's havebeen incorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al, Nucleic Acids Research, 2003, 21, 6365-6372).

In some embodiments, bicyclic nucleosides include, but are not limitedto, (A) a-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) beta-D-Methyleneoxy(4′-CH₂—O-2′) BNA, (C) Ethyleneoxy (4′-(CH₂)2-O-2′) BNA, (D) Aminooxy(4′-CH₂—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F),Methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio(4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, and (J) propylene carbocyclic(4′-(CH₂)₃-2′) BNA as depicted below

wherein Bx is the base moiety and R is, independently, H, a protectinggroup or C₁-C₂ alkyl, embodiments, bicyclic nucleoside having Formula I:

In certain emb

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)— is —CH₂—N(Rc)—CH₂—, —C(═O)—N(R_(c))—CH₂—,—CH₂—O—N(Rc)-, —CH₂—N(Rc)—O—, or —N(Rc)—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium.

In some embodiments, bicyclic nucleoside having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; Z_(a) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substitutedamide, thiol, or substituted thio.

In some embodiments, each of the substituted groups is, independently,mono or poly substituted with substituent groups independently selectedfrom halogen, oxo, hydroxyl, OJ_(c), NJ_(d), SJ_(C), N₃, OC(═X)J_(c),and NJ_(e)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d), and J_(e) is,independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O orNJ_(C).

In some embodiments, bicyclic nucleoside having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, orsubstituted acyl (C(═O)—).

In some embodiments, bicyclic nucleoside having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl;each q_(b), q_(c) and q_(d) is, independently, H, halogen, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C6 alkynyl, C₁-C₆ alkoxyl, substituted Q-C₆alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl, or substituted C₁-C₆aminoalkyl;

In some embodiments, bicyclic nucleoside having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; q_(a), q_(b), q_(c) and q_(f)are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substitutedC₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN,C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k),N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); orq_(e) and q_(f) together are ═C(q_(g))(q_(h)); q_(g) and q_(h) are each,independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNAmonomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine, anduracil, along with their oligomerization, and nucleic acid recognitionproperties have been described (see, e.g., Koshkin et al., Tetrahedron,1998, 54, 3607-3630). BNAs and preparation thereof are also described inWO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA, methyleneoxy (4′-CH₂—O-2′)BNA, and 2′-thio-BNAs, have also been prepared {see, e.g., Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of lockednucleoside analogs comprising oligodeoxyribonucleotide duplexes assubstrates for nucleic acid polymerases has also been described (see,e.g., Wengel et al., WO 99/14226). Furthermore, synthesis of2′-amino-BNA, a novel comformationally restricted high-affinityoligonucleotide analog, has been described in the art (see, e.g., Singhet al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino-and 2′-methylamino-BNA's have been prepared and the thermal stability oftheir duplexes with complementary RNA and DNA strands has beenpreviously reported.

In some embodiments, the bicyclic nucleoside has Formula VI:

In certain embodiments, bicyc

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, H, a hydroxyl protecting group,a conjugate group, a reactive phosphorus group, a phosphorus moiety, ora covalent attachment to a support medium; each qj, qj, q_(k) and ql is,independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl, substitutedC₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₂-C₁₂ alkoxyl, OJ_(j),SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j),C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k),N(H)C(═O)NJ_(j)J_(k), or (H)C(═S)NJ_(j)J_(k); and qi and q_(j) or ql andq_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each,independently, H, halogen, C₁-C₁₂ alkyl, or substituted C₁-C₆ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and thealkenyl analog, bridge 4′-CH═CH—CH₂-2′, have been described (see, e.g.,Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaeket al, J. Org. Chem., 2006, 71, 7731-77′40). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclicnucleoside” refers to a bicyclic nucleoside comprising a furanose ringcomprising a bridge connecting the 2′ carbon atom and the 4′ carbonatom.

As used herein, “monocylic nucleosides” refer to nucleosides comprisingmodified sugar moieties that are not bicyclic sugar moieties. In someembodiments, the sugar moiety, or sugar moiety analogue, of a nucleosidemay be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified atthe 2′ position. In some embodiments, such modifications includesubstituents selected from: a halide, including, but not limited tosubstituted and unsubstituted alkoxy, substituted and unsubstitutedthioalkyl, substituted and unsubstituted amino alkyl, substituted andunsubstituted alkyl, substituted and unsubstituted allyl, andsubstituted and unsubstituted alkynyl. In some embodiments, 2′modifications are selected from substituents including, but not limitedto: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂,OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]2, where n and m arefrom 1 to about 10. Other 2′-substituent groups can also be selectedfrom: C₁-C₁₂ alkyl; substituted alkyl; alkenyl; alkynyl; alkaryl;aralkyl; O-alkaryl or O-aralkyl; SH; SCH₃; OCN; Cl; Br; ON; CF₃; OCF₃;SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl;aminoalkylamino; polyalkylamino; substituted silyl; an R; a cleavinggroup; a reporter group; an intercalator; a group for improvingpharmacokinetic properties; and a group for improving thepharmacodynamic properties of an antisense compound, and othersubstituents having similar properties. In some embodiments, modifiednucleosides comprise a 2′-MOE side chain {see, e.g., Baker et al., J.Biol. Chem., 1997, 272, 1 1944-12000). Such 2′-MOE substitution havebeen described as having improved binding affinity compared tounmodified nucleosides and to other modified nucleosides, such as2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the2-MOE substituent also have been shown to be antisense inhibitors ofgene expression with promising features for in vivo use {see, e.g.,Martin, P., He/v. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia,1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24,630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16,917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THPnucleoside” means a nucleoside having a six-membered tetrahydropyran“sugar” substituted in for the pentofuranosyl residue in normalnucleosides (a sugar surrogate). Modified ?THP nucleosides include, butare not limited to, what is referred to in the art as hexitol nucleicacid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) {seeLeumann, C J. Bioorg. and Med. Chem. (2002) 10:841-854), fluoro HNA(F-HNA), or those compounds having Formula X:

X wherein independently for each of said at least one tetrahydropyrannucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the tetrahydropyran nucleoside analog to the antisense compoundor one of T₃ and T₄ is an internucleoside linking group linking thetetrahydropyran nucleoside analog to the antisense compound and theother of T₃ and T4 is H, a hydroxyl protecting group, a linked conjugategroup, or a 5′ or 3′-terminal group; q₁ q₂ q₃ q₄ q₅, q₆ and q₇ are each,independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, or substituted C₂-C₆ alkynyl;and one of R₁ and R₂ is hydrogen and the other is selected from halogen,substituted or unsubstituted alkoxy, NJ, J₂, SJ, N₃, OC(═X)J₁,OC(═X)NJ₁J₂, NJ₃C(═X) NJ₁J₂, and CN, wherein X is O, S, or NJ₁ and eachJ₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl.

In some embodiments, the modified THP nucleosides of Formula X areprovided wherein q_(m), q_(n), q_(p), q_(r), q_(s), q_(t), and q_(u) areeach H. In some embodiments, at least one of q_(m), q_(n), q_(p), q_(r),q_(s), q_(t) and q_(u) is other than H. In some embodiments, at leastone of q_(m), q_(n), q_(p), q_(r), q_(s), q_(t) and q_(u) is methyl. Insome embodiments, THP nucleosides of Formula X are provided wherein oneof R₁ and R₂ is F. In some embodiments, Riis fluoro and R₂ is H, R₁ ismethoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleosidecomprising a sugar comprising a substituent at the 2′ position otherthan H or OH. 2′-modified nucleosides, include, but are not limited tonucleosides with non-bridging 2′substituents, such as allyl, amino,azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂—O—CH₃,2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.2′-modified nucleosides may further comprise other modifications, forexample, at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a sugar comprising a fluoro group atthe 2′ position.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to anucleoside comprising a sugar comprising an —OCH₃ group at the 2′position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising aplurality of linked nucleosides.

In some embodiments, one or more of the plurality of nucleosides ismodified. In some embodiments, an oligonucleotide comprises one or moreribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds {see, e.g., review article:Leumann, J. C, Bioorganic and Medicinal Chemistry, 2002, 10, 841-854).Such ring systems can undergo various additional substitutions toenhance activity. Methods for the preparations of modified sugars arewell known to those skilled in the art. In nucleotides having modifiedsugar moieties, the nucleobase moieties (natural, modified, or acombination thereof) are maintained for hybridization with anappropriate nucleic acid target.

In some embodiments, antisense compounds comprise one or morenucleotides having modified sugar moieties. In some embodiments, themodified sugar moiety is 2′-MOE. In some embodiments, the 2′-MOEmodified nucleotides are arranged in a gapmer motif. In someembodiments, the modified sugar moiety is a cEt. In some embodiments,the cEt modified nucleotides are arranged throughout the wings of agapmer motif.

In some embodiments, in the BNA (LNA), R⁴* and R²* together designatethe biradical —O—CH(CH₂OCH₃)— (2′O-methoxyethyl bicyclic nucleicacid—Seth at al., 2010, J. Org. Chem)—in either the R- orS-configuration.

In some embodiments, in the BNA (LNA), R⁴* and R²* together designatethe biradical —O—CH(CH₂CH₃)— (2′O-ethyl bicyclic nucleic acid—Seth atal., 2010, J. Org. Chem).—in either the R- or S-configuration.

In some embodiments, in the BNA (LNA), R⁴* and R²* together designatethe biradical —O—CH(CH₃)—.—in either the R- or S-configuration. In someembodiments, R⁴* and R²* together designate the biradical—O—CH₂—O—CH₂——(Seth at al., 2010, J. Org. Chem).

In some embodiments, in the BNA (LNA), R⁴* and R²* together designatethe biradical —O—NR—CH₃——(Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA units have a structure selected from thefollowing group:

The oligomer may thus comprise or consist of a simple sequence ofnatural occurring nucleotides—preferably 2′-deoxynucleotides (referredto here generally as “DNA”), but also possibly ribonucleotides (referredto here generally as “RNA”), or a combination of such naturallyoccurring nucleotides and one or more non-naturally occurringnucleotides, i.e. nucleotide analogues. Such nucleotide analogues maysuitably enhance the affinity of the oligomer for the target sequence.

Incorporation of affinity-enhancing nucleotide analogues in theoligomer, such as BNA, (e.g.) LNA or 2′-substituted sugars, can allowthe size of the specifically binding oligomer to be reduced, and mayalso reduce the upper limit to the size of the oligomer beforenon-specific or aberrant binding takes place.

In some embodiments, the oligomer comprises at least 1 nucleosideanalogue. In some embodiments the oligomer comprises at least 2nucleotide analogues. In some embodiments, the oligomer comprises from3-8 nucleotide analogues, e.g. 6 or 7 nucleotide analogues. In the byfar most preferred embodiments, at least one of said nucleotideanalogues is a BNA, such as locked nucleic acid (LNA); for example atleast 3 or at least 4, or at least 5, or at least 6, or at least 7, or8, of the nucleotide analogues may be BNA, such as LNA. In someembodiments all the nucleotides analogues may be BNA, such as LNA.

It will be recognized that when referring to a preferred nucleotidesequence motif or nucleotide sequence, which consists of onlynucleotides, the oligomers of the invention which are defined by thatsequence may comprise a corresponding nucleotide analogue in place ofone or more of the nucleotides present in said sequence, such as BNAunits or other nucleotide analogues, which raise the duplexstability/T_(m) of the oligomer/target duplex (i.e. affinity enhancingnucleotide analogues).

A preferred nucleotide analogue is LNA, such as oxy-LNA (such asbeta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such asbeta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such asbeta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA andalpha-L-ENA). Most preferred is beta-D-oxy-LNA.

In some embodiments the nucleotide analogues present within the oligomerof the invention are independently selected from, for example:2′-O-alkyl-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, BNAunits, e.g. LNA units, arabino nucleic acid (ANA) units, 2′-fluoro-ANAunits, HNA units, INA (intercalating nucleic acid—Christensen, 2002.Nucl. Acids. Res. 2002 30: 4918-4925, hereby incorporated by reference)units and 2′MOE units. In some embodiments there is only one of theabove types of nucleotide analogues present in the oligomer of theinvention, such as the first region, or contiguous nucleotide sequencethereof.

In some embodiments the nucleotide analogues are 2′-O-methoxyethyl-RNA(2′MOE), 2′-fluoro-DNA monomers or LNA nucleotide analogues, and as suchthe oligonucleotide of the invention may comprise nucleotide analogueswhich are independently selected from these three types of analogue, ormay comprise only one type of analogue selected from the three types. Insome embodiments at least one of said nucleotide analogues is2′-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-MOE-RNA nucleotideunits. In some embodiments at least one of said nucleotide analogues is2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-fluoro-DNAnucleotide units.

In some embodiments, the oligomer according to the invention comprisesat least one BNA, e.g. Locked Nucleic Acid (LNA) unit, such as 1, 2, 3,4, 5, 6, 7, or 8 BNA/LNA units, such as from 3-7 or 4 to 8 BNA/LNAunits, or 3, 4, 5, 6 or 7 BNA/LNA units. In some embodiments, all thenucleotide analogues are BNA, such as LNA. In some embodiments, theoligomer may comprise both beta-D-oxy-LNA, and one or more of thefollowing LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in eitherthe beta-D or alpha-L configurations or combinations thereof. In someembodiments all BNA, such as LNA, cytosine units are 5′methyl-Cytosine.In some embodiments of the invention, the oligomer (such as the firstand optionally second regions) may comprise both BNA and LNA and DNAunits. In some embodiments, the combined total of LNA and DNA units is10-25, such as 10-24, preferably 10-20, such as 10-18, such as 12-16. Insome embodiments of the invention, the nucleotide sequence of theoligomer, of first region thereof, such as the contiguous nucleotidesequence consists of at least one BNA, e.g. LNA and the remainingnucleotide units are DNA units. In some embodiments the oligomer, orfirst region thereof, comprises only BNA, e.g. LNA, nucleotide analoguesand naturally occurring nucleotides (such as RNA or DNA, most preferablyDNA nucleotides), optionally with modified internucleotide linkages suchas phosphorothioate.

The term “nucleobase” refers to the base moiety of a nucleotide andcovers both naturally occurring a well as non-naturally occurringvariants. Thus, “nucleobase” covers not only the known purine andpyrimidine heterocycles but also heterocyclic analogues and tautomeresthereof. It will be recognized that the DNA or RNA nucleosides of regionB may have a naturally occurring and/or non-naturally occurringnucleobase(s).

Examples of nucleobases include, but are not limited to adenine,guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine. In some embodiments the nucleobases may beindependently selected from the group consisting of adenine, guanine,cytosine, thymidine, uracil, 5-methylcytosine. In some embodiments thenucleobases may be independently selected from the group consisting ofadenine, guanine, cytosine, thymidine, and 5-methylcytosine.

In some embodiments, at least one of the nucleobases present in theoligomer is a modified nucleobase selected from the group consisting of5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

LNA

The term “LNA” refers to a bicyclic nucleoside analogue which comprisesa C2*-C4* biradical (a bridge), and is known as “Locked Nucleic Acid”.It may refer to an LNA monomer, or, when used in the context of an “LNAoligonucleotide”, LNA refers to an oligonucleotide containing one ormore such bicyclic nucleotide analogues. In some aspects bicyclicnucleoside analogues are LNA nucleotides, and these terms may thereforebe used interchangeably, and is such embodiments, both are becharacterized by the presence of a linker group (such as a bridge)between C2′ and C4′ of the ribose sugar ring.

In some embodiments the LNA used in the oligonucleotide compounds of theinvention preferably has the structure of the general formula II:

wherein Y is selected from the group consisting of —O—, —CH₂O—, —S—,—NH—, N(R^(e)) and/or —CH₂—; Z and Z* are independently selected amongan internucleotide linkage, R^(H), a terminal group or a protectinggroup; B constitutes a natural or non-natural nucleotide base moiety(nucleobase), and R^(H) is selected from hydrogen and C₁₋₄-alkyl; R^(a),R^(b) R^(c), R^(d) and R^(e) are, optionally independently, selectedfrom the group consisting of hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂);and R^(H) is selected from hydrogen and C₁₋₄-alkyl. In some embodimentsR^(a), R^(b) R^(c), R^(d) and R^(e) are, optionally independently,selected from the group consisting of hydrogen and C₁₋₆ alkyl, such asmethyl. For all chiral centers, asymmetric groups may be found in eitherR or S orientation, for example, two exemplary stereochemical isomersinclude the beta-D and alpha-L isoforms, which may be illustrated asfollows:

Specific exemplary LNA units are shown below:

The term “thio-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and—CH₂—N(R)— where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which Y in thegeneral formula above represents —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B). R^(e) is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

RNAse Recruitment

It is recognized that an oligomeric compound may function via non RNasemediated degradation of target mRNA, such as by steric hindrance oftranslation, or other methods, In some embodiments, the oligomers of theinvention are capable of recruiting an endoribonuclease (RNase), such asRNase H.

It is preferable such oligomers, such as region A, or contiguousnucleotide sequence, comprises of a region of at least 6, such as atleast 7 consecutive nucleotide units, such as at least 8 or at least 9consecutive nucleotide units (residues), including 7, 8, 9, 10, 11, 12,13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplexwith the complementary target RNA is capable of recruiting RNase. Thecontiguous sequence which is capable of recruiting RNAse may be regionY′ as referred to in the context of a gapmer as described herein. Insome embodiments the size of the contiguous sequence which is capable ofrecruiting RNAse, such as region Y′, may be higher, such as 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability to recruit RNaseH. A oligomeris deemed capable of recruiting RNase H if, when provided with thecomplementary RNA target, it has an initial rate, as measured inpmol/l/min, of at least 1%, such as at least 5%, such as at least 10%or, more than 20% of the of the initial rate determined using DNA onlyoligonucleotide, having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Example 91-95 of EP 1 222 309.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when provided with the complementary RNA target,and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is lessthan 1%, such as less than 5%, such as less than 10% or less than 20% ofthe initial rate determined using the equivalent DNA onlyoligonucleotide, with no 2′ substitutions, with phosphorothioate linkagegroups between all nucleotides in the oligonucleotide, using themethodology provided by Example 91-95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when provided with the complementary RNA target, and RNaseH, theRNaseH initial rate, as measured in pmol/l/min, is at least 20%, such asat least 40%, such as at least 60%, such as at least 80% of the initialrate determined using the equivalent DNA only oligonucleotide, with no2′ substitutions, with phosphorothioate linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExample 91-95 of EP 1 222 309.

Typically the region of the oligomer which forms the consecutivenucleotide units which, when formed in a duplex with the complementarytarget RNA is capable of recruiting RNase consists of nucleotide unitswhich form a DNA/RNA like duplex with the RNA target. The oligomer ofthe invention, such as the first region, may comprise a nucleotidesequence which comprises both nucleotides and nucleotide analogues, andmay be e.g. in the form of a gapmer, a headmer or a mixmer.

A “headmer” is defined as an oligomer that comprises a region X′ and aregion Y′ that is contiguous thereto, with the 5′-most monomer of regionY′ linked to the 3′-most monomer of region X′. Region X′ comprises acontiguous stretch of non-RNase recruiting nucleoside analogues andregion Y′ comprises a contiguous stretch (such as at least 7 contiguousmonomers) of DNA monomers or nucleoside analogue monomers recognizableand cleavable by the RNase.

A “tailmer” is defined as an oligomer that comprises a region X′ and aregion Y′ that is contiguous thereto, with the 5′-most monomer of regionY′ linked to the 3′-most monomer of the region X′. Region X′ comprises acontiguous stretch (such as at least 7 contiguous monomers) of DNAmonomers or nucleoside analogue monomers recognizable and cleavable bythe RNase, and region X′ comprises a contiguous stretch of non-RNaserecruiting nucleoside analogues.

Other “chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA monomers or nucleoside analogue monomersrecognizable and cleavable by RNase, and (ii) non-RNase recruitingnucleoside analogue monomers.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogues also mediate RNase(e.g., RNaseH) binding and cleavage. Since α-L-LNA (BNA) monomersrecruit RNaseH activity to a certain extent, in some embodiments, gapregions (e.g., region Y′ as referred to herein) of oligomers containingα-L-LNA monomers consist of fewer monomers recognizable and cleavable bythe RNaseH, and more flexibility in the mixmer construction isintroduced.

Gapmer Design

In some embodiments, the oligomer of the invention, such as the firstregion, comprises or is a gapmer. A gapmer oligomer is an oligomer whichcomprises a contiguous stretch of nucleotides which is capable ofrecruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7DNA nucleotides, referred to herein in as region Y′ (Y′), wherein regionY′ is flanked both 5′ and 3′ by regions of affinity enhancing nucleotideanalogues, such as from 1-6 nucleotide analogues 5′ and 3′ to thecontiguous stretch of nucleotides which is capable of recruitingRNAse—these regions are referred to as regions X′ (X′) and Z′ (Z′)respectively. Examples of gapmers are disclosed in WO2004/046160,WO2008/113832, and WO2007/146511.

In some embodiments, the monomers which are capable of recruiting RNAseare selected from the group consisting of DNA monomers, alpha-L-LNAmonomers, C4′ alkylayted DNA monomers (see PCT/EP2009/050349 and Vesteret al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, herebyincorporated by reference), and UNA (unlinked nucleic acid) nucleotides(see Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporatedby reference). UNA is unlocked nucleic acid, typically where the C2-C3C—C bond of the ribose has been removed, forming an unlocked “sugar”residue. Preferably the gapmer comprises a (poly)nucleotide sequence offormula (5′ to 3′), X′—Y′—Z′, wherein; region X′ (X′) (5′ region)consists or comprises of at least one nucleotide analogue, such as atleast one BNA (e.g. LNA) unit, such as from 1-6 nucleotide analogues,such as BNA (e.g. LNA) units, and; region Y′ (Y′) consists or comprisesof at least five consecutive nucleotides which are capable of recruitingRNAse (when formed in a duplex with a complementary RNA molecule, suchas the mRNA target), such as DNA nucleotides, and; region Z′ (Z′)(3′region) consists or comprises of at least one nucleotide analogue,such as at least one BNA (e.g LNA unit), such as from 1-6 nucleotideanalogues, such as BNA (e.g. LNA) units.

In some embodiments, region X′ consists of 1, 2, 3, 4, 5 or 6 nucleotideanalogues, such as BNA (e.g. LNA) units, such as from 2-5 nucleotideanalogues, such as 2-5 LNA units, such as 3 or 4 nucleotide analogues,such as 3 or 4 LNA units; and/or region Z′ consists of 1, 2, 3, 4, 5 or6 nucleotide analogues, such as BNA (e.g. LNA) units, such as from 2-5nucleotide analogues, such as 2-5 BNA (e.g. LNA units), such as 3 or 4nucleotide analogues, such as 3 or 4 BNA (e.g. LNA) units.

In some embodiments Y′ consists or comprises of 5, 6, 7, 8, 9, 10, 11 or12 consecutive nucleotides which are capable of recruiting RNAse, orfrom 6-10, or from 7-9, such as 8 consecutive nucleotides which arecapable of recruiting RNAse. In some embodiments region Y′ consists orcomprises at least one DNA nucleotide unit, such as 1-12 DNA units,preferably from 4-12 DNA units, more preferably from 6-10 DNA units,such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments region X′ consist of 3 or 4 nucleotide analogues,such as BNA (e.g. LNA), region X′ consists of 7, 8, 9 or 10 DNA units,and region Z′ consists of 3 or 4 nucleotide analogues, such as BNA (e.g.LNA). Such designs include (X′—Y′—Z′) 3-10-3, 3-10-4, 4-10-3, 3-9-3,3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference. WO2008/113832, which claims priority fromU.S. provisional application 60/977,409 hereby incorporated byreference, refers to ‘shortmer’ gapmer oligomers. In some embodiments,oligomers presented here may be such shortmer gapmers.

In some embodiments the oligomer, e.g. region X′, is consisting of acontiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14nucleotide units, wherein the contiguous nucleotide sequence comprisesor is of formula (5′-3′), X′—Y′—Z′ wherein; X′ consists of 1, 2 or 3nucleotide analogue units, such as BNA (e.g. LNA) units; Y′ consists of7, 8 or 9 contiguous nucleotide units which are capable of recruitingRNAse when formed in a duplex with a complementary RNA molecule (such asa mRNA target); and Z′ consists of 1, 2 or 3 nucleotide analogue units,such as BNA (e.g. LNA) units.

In some embodiments X′ consists of 1 BNA (e.g. LNA) unit. In someembodiments X′ consists of 2 BNA (e.g. LNA) units. In some embodimentsX′ consists of 3 BNA (e.g. LNA) units. In some embodiments Z′ consistsof 1 BNA (e.g. LNA) units. In some embodiments Z′ consists of 2 BNA(e.g. LNA) units. In some embodiments Z′ consists of 3 BNA (e.g. LNA)units. In some embodiments Y′ consists of 7 nucleotide units. In someembodiments Y′ consists of 8 nucleotide units. In some embodiments Y′consists of 9 nucleotide units. In certain embodiments, region Y′consists of 10 nucleoside monomers. In certain embodiments, region Y′consists or comprises 1-10 DNA monomers. In some embodiments Y′comprises of from 1-9 DNA units, such as 2, 3, 4, 5, 6, 7, 8 or 9 DNAunits. In some embodiments Y′ consists of DNA units. In some embodimentsY′ comprises of at least one BNA unit which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in thealpha-L-configuration. In some embodiments Y′ comprises of at least onealpha-L-oxy BNA/LNA unit or wherein all the LNA units in thealpha-L-configuration are alpha-L-oxy LNA units. In some embodiments thenumber of nucleotides present in X′—Y′—Z′ are selected from the groupconsisting of (nucleotide analogue units-region Y′-nucleotide analogueunits): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2,1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3,3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1.In some embodiments the number of nucleotides in X′—Y′—Z′ are selectedfrom the group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2,3-7-4, and 4-7-3. In certain embodiments, each of regions X′ and Y′consists of three BNA (e.g. LNA) monomers, and region Y′ consists of 8or 9 or 10 nucleoside monomers, preferably DNA monomers. In someembodiments both X′ and Z′ consists of two BNA (e.g. LNA) units each,and Y′ consists of 8 or 9 nucleotide units, preferably DNA units. Invarious embodiments, other gapmer designs include those where regions X′and/or Z′ consists of 3, 4, 5 or 6 nucleoside analogues, such asmonomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) ormonomers containing a 2′-fluoro-deoxyribose sugar, and region Y′consists of 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, whereregions X′—Y′—Z′ have 3-9-3, 3-10-3, 5-10-5 or 4-12-4 monomers. Furthergapmer designs are disclosed in WO 2007/146511A2, hereby incorporated byreference.

Splice Switching Oligomers

In some embodiments, the antisense oligonucleotide is a splice switchingoligomer—i.e. an oligomer which targets the pre-mRNA causing analternative splicing of the pre-mRNA.

Targets for the splice switching oligomer may include TNF receptor, forexample the SSO may be one or more of the TNFR SSOs disclosed inWO2007/058894, WO08051306 A1 and PCT/EP2007/061211, hereby incorporatedby reference.

Splice switching oligomers are typically (essentially) not capable ofrecruiting RNaseH and as such gapmer, tailmer or headmer designs aregenerally not desirable. However, mixmer and totalmers designs aresuitable designs for SSOs.

Spice switching oligomers have also been used to target dystrophindeficiency in Duchenne muscular dystrophy.

Mixmers

Most antisense oligonucleotides are compounds which are designed torecruit RNase enzymes (such as RNaseH) to degrade their intended target.Such compounds include DNA phosphorothioate oligonucleotides and gapmer,headmers and tailmers. These compounds typically comprise a region of atleast 5 or 6 DNA nucleotides, and in the case of gapmers are flanked oneither side by affinity enhancing nucleotide analogues.

The oligomers of the present invention may operate via an RNase (such asRNaseH) independent mechanism. Examples of oligomers which operate via anon-RNaseH (or non-RNase) mechanism are mixmers and totalmers.

The term ‘mixmer’ refers to oligomers which comprise both naturally andnon-naturally occurring nucleotides, where, as opposed to gapmers,tailmers, and headmers there is no contiguous sequence of more than 5,and in some embodiments no more than 4 consecutive, such as no more thanthree consecutive, naturally occurring nucleotides, such as DNA units.In some embodiments, the mixmer does not comprise more than 5consecutive nucleoside analogues, such as BNA (LNA), and in someembodiments no more than 4 consecutive, such as no more than threeconsecutive, consecutive nucleoside analogues, such as BNA (LNA). Insuch mixmers the remaining nucleosides may, for example by DNAnucleosides, and/or in non-bicyclic nucleoside analogues, such as thosereferred to herein, for example, 2′ substituted nucleoside analogues,such as 2′-O-MOE and or 2′fluoro.

The oligomer according to the invention maybe mixmers—indeed variousmixmer designs are highly effective as oligomer or first region thereof,particularly when targeting microRNA (antimiRs), microRNA binding siteson mRNAs (Blockmirs) or as splice switching oligomers (SSOs). See forexample WO2007/112754 (LNA-AntimiRs™), WO2008/131807 (LNA spliceswitching oligos),

In some embodiments, the oligomer or mixmer may comprise of BNA and 2′substituted nucleoside analogues, optionally with DNA nucleosides—seefor example see WO07027894 and WO2007/112754 which are herebyincorporated by reference. Specific examples include oligomers or firstregions which comprise LNA, 2′-O-MOE and DNA, LNA, 2′fluoro and2′-O-MOE, 2′-O-MOE and 2′fluoro, 2′-O-MOE and 2′fluoro and LNA, or LNAand 2′-O-MOE and LNA and DNA.

In some embodiments, the oligomer or mixmer comprises or consists of acontiguous nucleotide sequence of repeating pattern of nucleotideanalogue and naturally occurring nucleotides, or one type of nucleotideanalogue and a second type of nucleotide analogues. The repeatingpattern, may, for instance be every second or every third nucleotide isa nucleotide analogue, such as BNA (LNA), and the remaining nucleotidesare naturally occurring nucleotides, such as DNA, or are a 2′substitutednucleotide analogue such as 2′MOE of 2′fluoro analogues as referred toherein, or, in some embodiments selected form the groups of nucleotideanalogues referred to herein. It is recognized that the repeatingpattern of nucleotide analogues, such as LNA units, may be combined withnucleotide analogues at fixed positions—e.g. at the 5′ or 3′ termini.

In some embodiments the first nucleotide of the oligomer or mixmer,counting from the 3′ end, is a nucleotide analogue, such as an LNAnucleotide.

In some embodiments, which maybe the same or different, the secondnucleotide of oligomer or mixmer, counting from the 3′ end, is anucleotide analogue, such as an LNA nucleotide.

In some embodiments, which maybe the same or different, the seventhand/or eighth nucleotide of oligomer or mixmer, counting from the 3′end, are nucleotide analogues, such as LNA nucleotides.

In some embodiments, which maybe the same or different, the ninth and/orthe tenth nucleotides of the first and/or second oligomer, counting fromthe 3′ end, are nucleotide analogues, such as LNA nucleotides.

In some embodiments, which maybe the same or different, the 5′ terminalof oligomer or mixmer is a nucleotide analogue, such as an LNAnucleotide.

The above design features may, in some embodiments be incorporated intothe mixmer design, such as antimiR mixmers.

In some embodiments, the oligomer or mixmer does not comprise a regionof more than 4 consecutive DNA nucleotide units or 3 consecutive DNAnucleotide units. In some embodiments, the mixmer does not comprise aregion of more than 2 consecutive DNA nucleotide units.

In some embodiments, the oligomer or mixmer comprises at least a regionconsisting of at least two consecutive nucleotide analogue units, suchas at least two consecutive LNA units.

In some embodiments, the oligomer or mixmer comprises at least a regionconsisting of at least three consecutive nucleotide analogue units, suchas at least three consecutive LNA units.

In some embodiments, the oligomer or mixmer of the invention does notcomprise a region of more than 7 consecutive nucleotide analogue units,such as LNA units. In some embodiments, the oligomer or mixmer of theinvention does not comprise a region of more than 6 consecutivenucleotide analogue units, such as LNA units. In some embodiments, theoligomer or mixmer of the invention does not comprise a region of morethan 5 consecutive nucleotide analogue units, such as LNA units. In someembodiments, the oligomer or mixmer of the invention does not comprise aregion of more than 4 consecutive nucleotide analogue units, such as LNAunits. In some embodiments, the oligomer or mixmer of the invention doesnot comprise a region of more than 3 consecutive nucleotide analogueunits, such as LNA units. In some embodiments, the oligomer or mixmer ofthe invention does not comprise a region of more than 2 consecutivenucleotide analogue units, such as LNA units. A mixmer is a oligomerwhich may comprise one or more short regions of DNA of no more than 4consecutive DNA nucleotides, and typically comprises alternating regionsof a nucleotide analogue (such as LNA units) and DNA nucleotides,optionally regions of other nucleotide analogues (e.g. non-LNAnucleotide analogues). Totalmers comprise of no DNA or RNA nucleotides(although may comprise analogues or derivatives of DNA and RNA).

In some embodiments, the oligomer (e.g. region A) of the invention may,in some embodiments, comprise of no more than 4 consecutive DNAnucleotides, or no more than 3 consecutive DNA nucleotides.

The following embodiments may apply to mixmers or totalmer oligomers(e.g. as region A): The oligomer (e.g. region A) of the invention may,in some embodiments, comprise of at least two alternating regions of LNAand non-LNA nucleotides (such as DNA or 2′ substituted nucleotideanalogues).

The oligomer of the invention may, in some embodiments, comprise acontiguous sequence of formula: 5′ ([LNA nucleotides]₁₋₅ and [non-LNAnucleotides]₁₋₄)₂₋₁₂. 3′.

In some embodiments, the 5′ nucleotide of the contiguous nucleotidesequence (or the oligomer) is an LNA nucleotide.

In some embodiments, the 3′ nucleotide of the contiguous nucleotidesequence is a nucleotide analogue, such as LNA, or the 2, 3, 4, 5 3′nucleotides are nucleotide analogues, such as LNA nucleotides, or othernucleotide analogues which confer enhanced serum stability to theoligomer.

In some embodiments, the contiguous nucleotide sequence of the oligomerhas a formula 5′ ([LNA nucleotides]₁₋₅-[non-LNAnucleotides]₁₋₄)₂₋₁₁-[LNA nucleotides]₁₋₅ 3′.

In some embodiments, the contiguous nucleotide sequence of the oligomerhas 2, 3 or 4 contiguous regions of LNA and non-LNA nucleotides—e.g.comprises formula 5′ ([LNA nucleotides]₁₋₅ and [non-LNAnucleotides]₁₋₄)₂₋₃, optionally with a further 3′ LNA region [LNAnucleotides]₁₋₅.

In some embodiments, the contiguous nucleotide sequence of the oligomercomprises 5′ ([LNA nucleotides]₁₋₃ and [non-LNA nucleotides]₁₋₃)₂₋₅,optionally with a further 3′ LNA region [LNA nucleotides]₁₋₃.

In some embodiments, the contiguous nucleotide sequence of the oligomercomprises 5′ ([LNA nucleotides]₁₋₃ and [non-LNA nucleotides]₁₋₃)₃,optionally with a further 3′ LNA region [LNA nucleotides]₁₋₃.

In some embodiments the non-LNA nucleotides are all DNA nucleotides.

In some embodiments, the non-LNA nucleotides are independently ordependently selected from the group consisting of DNA units, RNA units,2′-O-alkyl-RNA units, 2′-OMe-RNA units, 2′-amino-DNA units, and2′-fluoro-DNA units.

In some embodiments the non-LNA nucleotides are (optionallyindependently selected from the group consisting of 2′ substitutednucleoside analogues, such as (optionally independently) selected fromthe group consisting of 2′-O-alkyl-RNA units, 2′-OMe-RNA units,2′-amino-DNA units, 2′-AP, 2′-FANA, 2′-(3-hydroxy)propyl, and2′-fluoro-DNA units, and/or other (optionally) sugar modified nucleosideanalogues such as morpholino, peptide nucleic acid (PNA), CeNA, unlinkednucleic acid (UNA), hexitol nucleoic acid (HNA). bicyclo-HNA (see e.g.WO2009/100320), In some embodiments, the nucleoside analogues increasethe affinity of the first region for its target nucleic acid (or acomplementary DNA or RNA sequence). Various nucleoside analogues aredisclosed in Freier & Altmann; Nucl. Add Res., 1997, 25, 4429-4443 andUhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, herebyincorporated by reference.

In some embodiments, the non-LNA nucleotides are DNA nucleotides. Insome embodiments, the oligomer or contiguous nucleotide sequencecomprises of LNA nucleotides and optionally other nucleotide analogues(such as the nucleotide analogues listed under non-LNA nucleotides)which may be affinity enhancing nucleotide analogues and/or nucleotideanalogues which enhance serum stability.

In some embodiments, the oligomer or contiguous nucleotide sequencethereof consists of a contiguous nucleotide sequence of said nucleotideanalogues.

In some embodiments, the oligomer or contiguous nucleotide sequencethereof consists of a contiguous nucleotide sequence of LNA nucleotides.

In some embodiments, the oligomer or contiguous nucleotide sequence is8-12, such as 8-10, or 10-20, such as 12-18 or 14-16 nts in length.

In some embodiments, the oligomer or contiguous nucleotide sequence iscapable of forming a duplex with a complementary single stranded RNAnucleic acid molecule with phosphodiester internucleoside linkages,wherein the duplex has a T_(m) of at least about 60° C., such as atleast 65° C.

Example of a T_(m) Assay: The oligonucleotide: Oligonucleotide and RNAtarget (PO) duplexes are diluted to 3 mM in 500 ml RNase-free water andmixed with 500 ml 2×T_(m)-buffer (200 mM NaCl, 0.2 mM EDTA, 20 mMNaphosphate, pH 7.0). The solution is heated to 95° C. for 3 min andthen allowed to anneal in room temperature for 30 min. The duplexmelting temperatures (T_(m)) is measured on a Lambda 40 UVA/ISSpectrophotometer equipped with a Peltier temperature programmer PTP6using PE Templab software (Perkin Elmer). The temperature is ramped upfrom 20° C. to 95° C. and then down to 25° C., recording absorption at260 nm. First derivative and the local maximums of both the melting andannealing are used to assess the duplex T_(m).

Totalmers

A totalmer is a single stranded oligomer which only comprisesnon-naturally occurring nucleosides, such as sugar-modified nucleosideanalogues.

The first region according to the invention maybe totalmers—indeedvarious totalmer designs are highly effective as oligomers or firstregion thereof, e.g. particularly when targeting microRNA (antimiRs) oras splice switching oligomers (SSOs). In some embodiments, the totalmercomprises or consists of at least one XYX or YXY sequence motif, such asa repeated sequence XYX or YXY, wherein X is LNA and Y is an alternative(i.e. non LNA) nucleotide analogue, such as a 2′-O-MOE RNA unit and2′-fluoro DNA unit. The above sequence motif may, in some embodiments,be XXY, XYX, YXY or YYX for example.

In some embodiments, the totalmer may comprise or consist of acontiguous nucleotide sequence of between 7 and 16 nucleotides, such as9, 10, 11, 12, 13, 14, or 15 nucleotides, such as between 7 and 12nucleotides.

In some embodiments, the contiguous nucleotide sequence of the totalmercomprises of at least 30%, such as at least 40%, such as at least 50%,such as at least 60%, such as at least 70%, such as at least 80%, suchas at least 90%, such as 95%, such as 100% BNA (LNA) units. Theremaining units may be selected from the non-LNA nucleotide analoguesreferred to herein in, such those selected from the group consisting of2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′MOE RNA unit, orthe group 2′-OMe RNA unit and 2′-fluoro DNA unit.

In some embodiments the totalmer consist or comprises of a contiguousnucleotide sequence which consists only of LNA units. In someembodiments, the totalmer, such as the LNA totalmer, is between 7-12nucleoside units in length. In some embodiments, the totalmer (as theoligomer or first region thereof) may be targeted against a microRNA(i.e. be antimiRs)—as referred to WO2009/043353, which are herebyincorporated by reference. In some embodiments, the oligomer orcontiguous nucleotide sequence comprises of LNA nucleotides andoptionally other nucleotide analogues which may be affinity enhancingnucleotide analogues and/or nucleotide analogues which enhance serumstability.

In some embodiments, the oligomer or contiguous nucleotide sequencethereof consists of a contiguous nucleotide sequence of said nucleotideanalogues.

In some embodiments, the oligomer or contiguous nucleotide sequencethereof consists of a contiguous nucleotide sequence of LNA nucleotides.

MicroRNA Modulation Via the Oligomer or First Region Thereof.

In some embodiments, the oligomer or first region thereof is an antimiR,which comprises or consists of a contiguous nucleotide sequence which iscorresponds to or is fully complementary to a mature microRNA or partthereof. The use of the present invention in controlling the in vivoactivity of microRNA is considered of primary importance due to the factthat microRNAs typically regulate numerous mRNAs in the subject. Theability to inactivate therapeutic antimiRs is therefore very desirable.

Numerous microRNAs are related to a number of diseases. For example:non-limiting examples of therapeutic indications which may be treated bythe pharmaceutical compositions of the invention:

microRNA Possible medical indications miR-1 Cardiac arythmia miR-21Glioblastoma, breast cancer, hepatocellular carcinoma, colorectalcancer, sensitization of gliomas to cytotoxic drugs, cardiac hypertrophymiR-21, miR- Response to chemotherapy and regulation of 200b and miR-cholangiocarcinoma growth 141 miR-122 hypercholesterolemia, hepatitis Cinfection, hemochromatosis miR-19b lymphoma and other tumour typesmiR-26a Osteoblast differentiation of human stem cells miR-155 lymphoma,pancreatic tumor development, breast and lung cancer miR-203 PsoriasismiR-375 diabetes, metabolic disorders, glucose-induced insulin secretionfrom pancreatic endocrine cells miR-181 myoblast differentiation, autoimmune disorders miR-10b Breast cancer cell invasion and metastasismiR-125b-1 Breast, lung, ovarian and cervical cancer miR-221 and 222Prostate carcinoma, human thyroid papillary car, human hepatocellularcarcinoma miRNA-372 and - testicular germ cell tumors. 373 miR-142B-cell leukemia miR-17 - 19b B-cell lymphomas, lung cancer,hepatocellular cluster carcinoma

Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been indicated as atarget of miR-21. Myotrophin (mtpn) mRNA has been indicated as a targetof miR 375.

The oligomer or first region thereof may therefore be an antimir whichtargets (i.e. comprises or consists of a contiguous nucleotide sequencewhich is fully complementary to (a corresponding region of) one of themicroRNAs listed above or comprises of no more than a single mismatchthereto.

Hence, some aspects of the invention relates to the treatment of adisease associated with the expression of microRNAs selected from thegroup consisting of infectious diseases such as viral diseases such ashepatitis C virus and HIV, fragile X mental retardation, inflammatorydiseases, cancer, such as chronic lymphocytic leukemia, breast cancer,lung cancer and colon cancer.

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs thatact as post-transcriptional regulators of gene expression bybase-pairing with their target mRNAs. The mature miRNAs are processedsequentially from longer hairpin transcripts by the RNAse IIIribonucleases Drosha. Mature microRNAs (miRs) typically between 20 and25 contiguous RNA nucleotides. It is now widely established that severalmicroRNAs are associated with medical conditions and disease, andseveral companies are developing therapeutics based on oligomers whicheither mimic microRNAs or specifically hybridse to specific microRNAsassociated with disease phenotypes—such oligomers are referred to,herein, as microRNA mimics and antimiRs respectfully, and the oligomeror first region thereof, in some embodiments may be such microRNAmodulating oligomers.

In some embodiments the oligomer or first region thereof according tothe invention, consists or comprises of a contiguous nucleotide sequencewhich corresponds to or is fully complementary to a microRNA sequence,such as a mature microRNA sequence, such as the human microRNAspublished in miRBase(http://microrna.sanqer.ac.uk/cqi-bin/sequences/mirna_summary.pl?orq=hsa).In some embodiment the microRNA is a viral microRNA. At the time ofwriting, in miRbase 19, there are 1600 precursors and 2042 mature humanmiRNA sequences in miRBase which are all hereby incorporated byreference, including the mature microRNA sequence of each humanmicroRNA. Other human microRNAs which may be targeted by the oligomer orfirst region thereof include those disclosed in WO08040355A, herebyincorporated by reference. In some embodiments the oligomer or firstregion thereof according to the invention, consists or comprises of acontiguous nucleotide sequence which corresponds to or is fullycomplementary to a microRNA sequence selected from the group consistingof hsa-miR19b, hsa-miR21, hsa-miR 122, hsa-miR 142 a7b, hsa-miR 155, andhsa-miR 375. In some embodiments the oligomer or first region thereofaccording to the invention, consists or comprises of a contiguousnucleotide sequence which corresponds to or is fully complementary to amicroRNA sequence selected from the group consisting of hsa-miR221 andhsa-miR222. In some embodiments the oligomer or first region thereofaccording to the invention, consists or comprises of a contiguousnucleotide sequence which corresponds to or is fully complementary tohsa-miR122 (NR_029667.1 GI: 262205241), such as the mature has-miR-122.

In some embodiments when the oligomer or first region thereof targetsmiR-122, the oligomer is for the use in the treatment of hepatitis Cinfection.

AntimiR Oligomers

Preferred oligomer or first region thereof ‘antimiR’ designs andoligomers are disclosed in WO2007/112754, WO2007/112753,PCT/DK2008/000344 and U.S. provisional applications 60/979,217 and61/028,062, all of which are hereby incorporated by reference. In someembodiments, the oligomer or first region thereof is an antimiR which isa mixmer or a totalmer.

AntimiR oligomers are oligomers which consist or comprise of acontiguous nucleotide sequence which is fully complementary to, oressentially complementary to (i.e. may comprise one or two mismatches),to a microRNA sequence, or a corresponding sub-sequence thereof. In thisregards it is considered that the antimiR may be comprise a contiguousnucleotide sequence which is complementary or essentially complementaryto the entire mature microRNA, or the antimiR may be comprise acontiguous nucleotide sequence which is complementary or essentiallycomplementary to a sub-sequence of the mature microRNA orpre-microRNA—such a sub-sequence (and therefore the correspondingcontiguous nucleotide sequence) is typically at least 8 nucleotides inlength, such as between 8 and 25 nucleotides, such as 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 nucleotides in length, suchas between 10-17 or 10-16 nucleotides, such as between 12-15nucleotides.

Numerous designs of AntimiRs have been suggested, and typically antimiRsfor therapeutic use, such as the contiguous nucleotide sequence thereofcomprise one or more nucleotide analogues units.

In some embodiments the antimiR may have a gapmer structure as hereindescribed. However, as explained in WO2007/112754 and WO2007/112753,other designs may be preferable, such as mixmers, or totalmers.

WO2007/112754 and WO2007/112753, both hereby incorporated by reference,provide antimiR oligomers and antimiR oligomer designs where theoligomers which are complementary to mature microRNA

In some embodiments, a subsequence of the antimiR corresponds to themiRNA seed region. In some embodiments, the first or second 3′nucleobase of the oligomer corresponds to the second 5′ nucleotide ofthe microRNA sequence.

In some antimiR embodiments, nucleobase units 1 to 6 (inclusive) of theoligomer as measured from the 3′ end the region of the oligomer arecomplementary to the microRNA seed region sequence.

In some antimiR embodiments, nucleobase units 1 to 7 (inclusive) of theoligomer as measured from the 3′ end the region of the oligomer arecomplementary to the microRNA seed region sequence.

In some e antimiR embodiments, nucleobase units 2 to 7 (inclusive) ofthe oligomer as measured from the 3′ end the region of the oligomer arecomplementary to the microRNA seed region sequence.

In some embodiments, the antimiR oligomer comprises at least onenucleotide analogue unit, such as at least one LNA unit, in a positionwhich is within the region complementary to the miRNA seed region. TheantimiR oligomer may, in some embodiments comprise at between one and 6or between 1 and 7 nucleotide analogue units, such as between 1 and 6and 1 and 7 LNA units, in a position which is within the regioncomplementary to the miRNA seed region.

In some embodiments, the antimiR of the invention is 7, 8 or 9nucleotides long, and comprises a contiguous nucleotide sequence whichis complementary to a seed region of a human or viral microRNA, andwherein at least 80%, such as 85%, such as 90%, such as 95%, such as100% of the nucleotides are LNA.

In some embodiments, the antimiR of the invention is 7, 8 or 9nucleotides long, and comprises a contiguous nucleotide sequence whichis complementary to a seed region of a human or viral microRNA, andwherein at least 80% of the nucleotides are LNA, and wherein at least80%, such as 85%, such as 90%, such as 95%, such as 100% of theinternucleotide bonds are phosphorothioate bonds.

In some embodiments, the antimiR comprises one or two LNA units inpositions three to eight, counting from the 3′ end. This is consideredadvantageous for the stability of the A-helix formed by theoligonucleotide:microRNA duplex, a duplex resembling an RNA:RNA duplexin structure.

The table on pages 48 line 15 to page 51, line 9 of WO2007/112754provides examples of anti microRNA oligomers (i.e. antimiRs which may bethe oligomer or first region thereof) and is hereby specificallyincorporated by reference.

MicroRNA Mimics

In some embodiments the oligomer or first region thereof is in the formof a miRNA mimic which can be introduced into a cell to repress theexpression of one or more mRNA target(s). miRNA mimics are typicallyfully complementary to the full length miRNA sequence. miRNA mimics arecompounds comprising a contiguous nucleotide sequence which arehomologous to a corresponding region of one, or more, of the miRNAsequences provided or referenced to herein. The use of miRNA mimics orantimiRs can be used to (optionally) further repress the mRNA targets,or to silence (down-regulate) the miRNA, thereby inhibiting the functionof the endogenous miRNA, causing derepression and increased expressionof the mRNA target.

Aptamers

In some embodiments the oligomer or first region thereof may be atherapeutic aptamer, a spiegelmer. Please note that aptamers may also beligands, such as receptor ligands, and may therefore be used as atargeting moiety (i.e. region 3). Aptamers (also referred to asSpiegelmers) in the context of the present invention as nucleic acids ofbetween 20 and 50 nucleotides in length, which have been selected on thebasis of their conformational structure rather than the sequence ofnucleotides—they elicit their therapeutic effect by binding with atarget protein directly in vivo and they do not, therefore, comprise ofthe reverse complement of their target—indeed their target is not anucleic acid but a protein. Specific aptamers which may be the oligomeror first region thereof include Macugen (OSI Pharmaceuticals) orARC1779, (Archemix, Cambridge, Mass.). In some embodiments, the oligomeror first region thereof is not an aptamer. In some embodiments theoligomer or first region thereof is not an aptamer or a spiegelmer.

siRNA Complexes

In some embodiments, the oligomer or first region thereof may be part ofa siRNA complex—i.e. the antisense or passenger strand of the siRNAcomplex. An siRNA complex is capable of mediating RNA interference.

In some embodiments the siRNA complex comprises two single strandedoligomers of between 17-25 nts in length, such as 18, 19, 20, 21, 22,23, 24 nucleotides in length, such as between 21-23 nucleotides inlength. In some embodiments, the sense and/or antisense strand of thesiRNA may comprise a 3′ overhang, typically of 1, 2 or 3 nucleotides.Suitably, the sense and or antisense strand may comprise one or morenucleotide analogues.

In some embodiments the siRNA complex is a siLNA, such as the siRNAdesigns described in WO2004/000192, WO2005/073378, WO2007/085485 all ofwhich are hereby incorporated by reference. An siLNA is a siRNA whichcomprises at least one LNA unit.

In some embodiments, the siRNA complex is a sisiLNA, such as thosedescribed in WO2007/107162, hereby incorporated by reference. In someembodiments, the oligomer or first region thereof, of the invention isthe sense strand of the siRNA, and as such may be non-complementary tothe target (indeed, may be homologous to the intended target).

In some embodiments, the oligomer or compound of the invention is not asiRNA or a siLNA.

Internucleotide Linkages

The nucleoside monomers of the oligomers (e.g. first and second regions)described herein are coupled together via [internucleoside] linkagegroups. Suitably, each monomer is linked to the 3′ adjacent monomer viaa linkage group.

The person having ordinary skill in the art would understand that, inthe context of the present invention, the 5′ monomer at the end of anoligomer does not comprise a 5′ linkage group, although it may or maynot comprise a 5′ terminal group.

The terms “linkage group” or “internucleotide linkage” are intended tomean a group capable of covalently coupling together two nucleotides.Specific and preferred examples include phosphate groups andphosphorothioate groups.

The nucleotides of the oligomer of the invention or contiguousnucleotides sequence thereof are coupled together via linkage groups.Suitably each nucleotide is linked to the 3′ adjacent nucleotide via alinkage group.

Suitable internucleotide linkages include those listed withinWO2007/031091, for example the internucleotide linkages listed on thefirst paragraph of page 34 of WO2007/031091 (hereby incorporated byreference).

It is, in some embodiments, other than the phosphodiester linkage(s) orregion B, the preferred to modify the internucleotide linkage from itsnormal phosphodiester to one that is more resistant to nuclease attack,such as phosphorothioate or boranophosphate—these two, being cleavableby RNase H, also allow that route of antisense inhibition in reducingthe expression of the target gene.

Suitable sulphur (S) containing internucleotide linkages as providedherein may be preferred, such as phosphorothioate or phosphodithioate.Phosphorothioate internucleotide linkages are also preferred,particularly for the first region, such as in gapmers, mixmers, antimirssplice switching oligomers, and totalmers.

For gapmers, the internucleotide linkages in the oligomer may, forexample be phosphorothioate or boranophosphate so as to allow RNase Hcleavage of targeted RNA. Phosphorothioate is preferred, for improvednuclease resistance and other reasons, such as ease of manufacture.

In one aspect, with the exception of the phosphodiester linkage betweenthe first and second region, and optionally within region B, theremaining internucleoside linkages of the oligomer of the invention, thenucleotides and/or nucleotide analogues are linked to each other bymeans of phosphorothioate groups. In some embodiments, at least 50%,such as at least 70%, such as at least 80%, such as at least 90% such asall the internucleoside linkages between nucleosides in the first regionare other than phosphodiester (phosphate), such as are selected from thegroup consisting of phosphorothioate phosphorodithioate, orboranophosphate. In some embodiments, at least 50%, such as at least70%, such as at least 80%, such as at least 90% such as all theinternucleoside linkages between nucleosides in the first region arephosphorothioate.

WO09124238 refers to oligomeric compounds having at least one bicyclicnucleoside attached to the 3′ or 5′ termini by a neutral internucleosidelinkage. The oligomers of the invention may therefore have at least onebicyclic nucleoside attached to the 3′ or 5′ termini by a neutralinternucleoside linkage, such as one or more phosphotriester,methylphosphonate, MMI, amide-3, formacetal or thioformacetal. Theremaining linkages may be phosphorothioate.

Conjugates, Targeting Moieties and Blocking Groups

The term “conjugate” is intended to indicate a heterogenous moleculeformed by the covalent attachment (“conjugation”) of the oligomer asdescribed herein to one or more non-nucleotide, or non-polynucleotidemoieties. Examples of non-nucleotide or non-polynucleotide moietiesinclude macromolecular agents such as proteins, fatty acid chains, sugarresidues, glycoproteins, polymers, or combinations thereof. Typicallyproteins may be antibodies for a target protein. Typical polymers may bepolyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention maycomprise both a polynucleotide region which typically consists of acontiguous sequence of nucleotides, and a further non-nucleotide region.When referring to the oligomer of the invention consisting of acontiguous nucleotide sequence, the compound may comprise non-nucleotidecomponents, such as a conjugate component.

In various embodiments of the invention the oligomeric compound islinked to ligands/conjugates, which may be used, e.g. to increase thecellular uptake of oligomeric compounds. WO2007/031091 provides suitableligands and conjugates, which are hereby incorporated by reference.

In various embodiments where the compound of the invention consists of aspecified nucleic acid or nucleotide sequence, as herein disclosed, thecompound may also comprise at least one non-nucleotide ornon-polynucleotide moiety (e.g. not comprising one or more nucleotidesor nucleotide analogues) covalently attached to said compound.

In some embodiments, the conjugate may be a lipophilic conjugate or aproteins (e.g., antibodies, enzymes, serum proteins); peptides; vitamins(water-soluble or lipid-soluble); polymers (water-soluble orlipid-soluble); small molecules including drugs, toxins, reportermolecules, and receptor ligands; carbohydrate complexes; nucleic acidcleaving complexes; metal chelators (e.g., porphyrins, texaphyrins,crown ethers, etc.); intercalators including hybridphotonuclease/intercalators; crosslinking agents (e.g., photoactive,redox active), and combinations and derivatives thereof. Numeroussuitable conjugate moieties, their preparation and linkage to oligomericcompounds are provided, for example, in WO 93/07883 and U.S. Pat. No.6,395,492, each of which is incorporated herein by reference in itsentirety. Oligonucleotide conjugates and their syntheses are alsoreported in comprehensive reviews by Manoharan in Antisense DrugTechnology, Principles, Strategies, and Applications, S. T. Crooke, ed.,Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and NucleicAcid Drug Development, 2002, 12, 103, each of which is incorporatedherein by reference in its entirety.

Conjugation (to a conjugate moiety) may enhance the activity, cellulardistribution or cellular uptake of the oligomer of the invention. Suchmoieties include, but are not limited to, antibodies, polypeptides,lipid moieties such as a cholesterol moiety, cholic acid, a thioether,e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipids, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example from 1-50, such as 2-20 such as 3-10 amino acid residues inlength, and/or polyalkylene oxide such as polyethylglycol (PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference.

The use of a conjugate is often associated with enhanced pharmacokineticor pharmeodynamic dynamic properties. However, the presence of aconjugate group may interfere with the activity of the oligonucleotideagainst its intended target, for example via steric hindrance preventinghybridization or nuclease recruitment (e.g. RNAseH or RISC recruitment).The use of a DNA and/or RNA phosphodiester region (region B) between theoligonucleotide (region A) and the conjugate moiety (X), as according tothe present invention, allows for the improved properties due to thepresence of the conjugate group, whilst ensuring that once at the targettissue, the conjugate group does not prevent effective activity of theoligonucleotide.

The oligonucleotide of the invention is, in some embodiments, covalentlyattached to one or more conjugate group, optionally through one or morelinkers. The resulting conjugate compounds may, for example havemodified enhanced properties, such as modified or enhancedpharmacokinetic, pharmeodynamic, and other properties compared withnon-conjugated oligomeric compounds. A conjugate moiety that can modifyor enhance the pharmacokinetic properties of an oligomeric compound canimprove cellular distribution, bioavailability, metabolism, excretion,permeability, and/or cellular uptake of the oligomeric compound. Aconjugate moiety that can modify or enhance pharmacodynamic propertiesof an oligomeric compound can improve activity, resistance todegradation, sequence-specific hybridization, uptake, and the like. Insome embodiments, the conjugate group may reduce or prevent inappropriate activity of the oligonucleotide, e.g. off target activity oractivity in non-target tissues or organs. This may be achieved by use ofa blocking moiety, which may for example be a conjugate, the presence ofthe blocking group covalently attached to the oligonucleotide(optionally via a linker), may prevent or hinder oligonucleotidehybridization and/or activity. The cleavage of the DNA/RNAphosphodiester region (e.g. at the intended target site), removes theblocking group, allowing delivery of the active oligonucleotide at theintended site.

In some embodiments, the compound of the invention comprises a conjugategroup.

It will be recognized that one conjugate group may be used, for examplefor targeting to a specific tissue, for example a lipophilic group fortargeting to the liver, and a second conjugate group may be used toprovide a further benefit, for example a blocking group or a furthertherapeutic entity. Suitable one or both of the conjugates/moieties maybe linked to the oligonucleotide via the DNA/RNA phosphodiester regionaccording to the present invention. In some embodiments, the conjugateis covalently bound to the oligonucleotide, optionally via a linker, atthe 5′ and/or 3′ termini of the oligonucleotide. In this respect, if twoconjugate/moiety groups are used, one may be linked to the 5′ terminiand one to the 3′ termini.

Carbohydrate Conjugates

In some embodiments, the conjugate group is selected from the groupconsisting of a carbohydrate, a lipophilic moiety, a polymer, a proteinor peptide, a label or dye, a small molecule, such as a small moleculetherapeutic moiety, a cell surface receptor ligand.

In some embodiments, the conjugate is or may comprise a carbohydrate orcomprises a carbohydrate group. In some embodiments, the carbohydrate isselected from the group consisting of galactose, lactose,n-acetylgalactosamine, mannose, and mannose-6-phosphate. In someembodiments, the conjugate group is or may comprise mannose ormannose-6-phosphate. Carbohydrate conjugates may be used to enhancedelivery or activity in a range of tissues, such as liver and/or muscle.See, for example, EP1495769, WO99/65925, Yang et al., Bioconjug Chem(2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

In some embodiments, the conjugate group is a carbohydrate moiety. Inaddition, the oligomer may further comprise one or more additionalconjugate moieties, of which lipophilic or hydrophobic moieties areparticularly interesting. These may for example, act as pharmacokineticmodulators, and may be covalently linked to either the carbohydrateconjugate, a linker linking the carbohydrate conjugate to the oligomeror a linker linking multiple carbohydrate conjugates (multi-valent)conjugates, or to the oligomer, optionally via a linker, such as a biocleavable linker. I

In some embodiments, the conjugate is or may comprise a carbohydrate orcomprises a carbohydrate group. In some embodiments, the carbohydrate isselected from the group consisting of galactose, lactose,n-acetylgalactosamine, mannose, and mannose-6-phosphate. In someembodiments, the conjugate group is or may comprise mannose ormannose-6-phosphate. Carbohydrate conjugates may be used to enhancedelivery or activity in a range of tissues, such as liver and/or muscle.See, for example, EP1495769, WO99/65925, Yang et al., Bioconjug Chem(2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

GalNAc Conjugates

The invention also provides oligonucleotides, such as LNA antisenseoligomers, which are conjugated to an asialoglycoprotein receptortargeting moiety. In some embodiments, the conjugate moiety (such as thethird region or region C) comprises an asialoglycoprotein receptortargeting moiety, such as galactose, galactosamine,N-formyl-galactosamine, Nacetylgalactosamine, N-propionyl-galactosamine,N-n-butanoyl-galactosamine, and N-isobutanoylgalactos-amine. In someembodiments the conjugate comprises a galactose cluster, such asN-acetylgalactosamine trimer. In some embodiments, the conjugate moietycomprises a GalNAc (N-acetylgalactosamine), such as a mono-valent,di-valent, tri-valent of tetra-valent GalNAc. Trivalent GalNAcconjugates may be used to target the compound to the liver. GalNAcconjugates have been used with methylphosphonate and PNA antisenseoligonucleotides (e.g. U.S. Pat. No. 5,994,517 and Hangeland et al.,Bioconjug Chem. 1995 November-December; 6(6):695-701) and siRNAs (e.g.WO2009/126933, WO2012/089352 & WO2012/083046). The GalNAc references andthe specific conjugates used therein are hereby incorporated byreference. WO2012/083046 discloses siRNAs with GalNAc conjugate moietieswhich comprise cleavable pharmacokinetic modulators, which are suitablefor use in the present invention, the preferred pharmacokineticmodulators are C16 hydrophobic groups such as palmitoyl,hexadec-8-enoyl, oleyl, (9E, 12E)-octadeca-9,12-dienoyl, dioctanoyl, andC16-C20 acyl. The '046 cleavable pharmacokinetic modulators may also becholesterol.

The ‘targeting moieties (conjugate moieties) may be selected from thegroup consisting of: galactose, galactosamine, N-formyl-galactosamine,N-acetylgalactosamine, Npropionyl-galactosamine,N-n-butanoyl-galactosamine, N-iso-butanoylgalactos-amine, galactosecluster, and N-acetylgalactosamine trimer and may have a pharmacokineticmodulator selected from the group consisting of: hydrophobic grouphaving 16 or more carbon atoms, hydrophobic group having 16-20 carbonatoms, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl,dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clustersdisclosed in '046 include: (E)-hexadec-8-enoyl (C16), oleyl (C₁₈),(9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl(C12), C-20 acyl, C24 acyl, dioctanoyl (2×C8). The targetingmoiety-pharmacokinetic modulator targeting moiety may be linked to thepolynucleotide via a physiologically labile bond or, e.g. a disulfidebond, or a PEG linker. The invention also relates to the use ofphospodiester linkers between the oligomer and the conjugate group(these are referred to as region B herein, and suitably are positionedbetween the LNA oligomer and the carbohydrate conjugate group).

For targeting hepatocytes in liver, a preferred targeting ligand is agalactose cluster.

A galactose cluster comprises a molecule having e.g. comprising two tofour terminal galactose derivatives. As used herein, the term galactosederivative includes both galactose and derivatives of galactose havingaffinity for the asialoglycoprotein receptor equal to or greater thanthat of galactose. A terminal galactose derivative is attached to amolecule through its C—I carbon. The asialoglycoprotein receptor (ASGPr)is unique to hepatocytes and binds branched galactose-terminalglycoproteins. A preferred galactose cluster has three terminalgalactosamines or galactosamine derivatives each having affinity for theasialoglycoprotein receptor. A more preferred galactose cluster hasthree terminal N-acetyl-galactosamines. Other terms common in the artinclude tri-antennary galactose, tri-valent galactose and galactosetrimer. It is known that tri-antennary galactose derivative clusters arebound to the ASGPr with greater affinity than bi-antennary ormono-antennary galactose derivative structures (Baenziger and Fiete,1980, Cell, 22, 611-620; Connolly et al., 1982, 1. Biol. Chern., 257,939-945). Multivalency is required to achieve nM affinity. According toWO 2012/083046 the attachment of a single galactose derivative havingaffinity for the asialoglycoprotein receptor does not enable functionaldelivery of the RNAi polynucleotide to hepatocytes in vivo whenco-administered with the delivery polymer.

A galactose cluster may comprise two or preferably three galactosederivatives each linked to a central branch point. The galactosederivatives are attached to the central branch point through the C—Icarbons of the saccharides. The galactose derivative is preferablylinked to the branch point via linkers or spacers (which may be regionY). A preferred spacer is a flexible hydrophilic spacer (U.S. Pat. No.5,885,968; Biessen et al. J. Med. Chern. 1995 Vol. 39 p. 1538-1546). Apreferred flexible hydrophilic spacer is a PEG spacer. A preferred PEGspacer is a PEG3 spacer. The branch point can be any small moleculewhich permits attachment of the three galactose derivatives and furtherpermits attachment of the branch point to the oligomer. An exemplarybranch point group is a di-lysine. A di-lysine molecule contains threeamine groups through which three galactose derivatives may be attachedand a carboxyl reactive group through which the di-lysine may beattached to the oligomer. Attachment of the branch point to oligomer mayoccur through a linker or spacer. A preferred spacer is a flexiblehydrophilic spacer. A preferred flexible hydrophilic spacer is a PEGspacer. A preferred PEG spacer is a PEG3 spacer (three ethylene units).The galactose cluster may be attached to the 3′ or 5′ end of theoligomer using methods known in the art.

A preferred galactose derivative is an N-acetyl-galactosamine (GalNAc).Other saccharides having affinity for the asialoglycoprotein receptormay be selected from the list comprising: galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. Theaffinities of numerous galactose derivatives for the asialoglycoproteinreceptor have been studied (see for example: Jobst, S. T. and Drickamer,K. JB. C. 1996, 271, 6686) or are readily determined using methodstypical in the art.

A GalNac conjugate is illustrated in FIG. 1. Further examples of theconjugate of the invention are depicted in FIG. 26 where A may, forexample, be an LNA antisense olibonucleotide.

As described herein, a carbohydrate conjugate (e.g. GalNAc) maytherefore be linked to the oligomer via a biocleavable linker, such asregion B as defined herein, and optionally region Y, which isillustrated as a di-lysine in the above diagrams.

Where at the hydrophobic or lipophilic (or further conjugate) moiety(i.e. pharmacokinetic modulator) in the above GalNac cluster conjugatesis, when using BNA or LNA oligomers, such as LNA antisenseoligonucleotides, optional.

See the figures for specific GalNac clusters used in the present study,Conj 1, 2, 3, 4 and Conj1a, 2a, 3a and 4a (which are shown with anoptional C6 linker which joins the GalNac cluster to the oligomer—SeeFIGS. 12 and 17).

Each carbohydrate moiety of a GalNac cluster (e.g. GalNAc) may thereforebe joined to the oligomer via a spacer, such as (poly)ethylene glycollinker (PEG), such as a di, tri, tetra, penta, hexa-ethylene glycollinker. As is shown above the PEG moiety forms a spacer between thegalactose sugar moiety and a peptide (trilysine is shown) linker.

In some embodiments, the GalNac cluster comprises a peptide linker, e.g.a Tyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide, which is attached tothe oligomer (or to region Y or region B) via a biradical linker, forexample the GalNac cluster may comprise the following biradical linkers:

R¹ is a biradical preferably selected from —C₂H₄—, —C₃H₆—, —C₄H₈—,—C₅H₁₀—, —C₆H₁₂—, 1,4-cyclohexyl (—C₆H₁₀—), 1,4-phenyl (—C₆H₄—),—C₂H₄OC₂H₄—, —C₂H₄(OC₂H₄)₂— or —C₂H₄(OC₂H₄)₃—. In addition, thecarbohydrate conjugate (e.g. GalNAc), or carbohydrate-linker moiety(e.g. carbohydrate-PEG moiety) may be covalently joined (linked) to theoligomer (or region B) via a branch point group such as, an amino acid,or peptide, which suitably comprises two or more amino groups (such as3, 4, or 5), such as lysine, di-lysine or tri-lysine ortetra-lysine. Atri-lysine molecule contains four amine groups through which threecarbohydrate conjugate groups, such as galactose & derivatives (e.g.GalNAc) and a further conjugate such as a hydrophobic or lipophilicmoiety/group may be attached and a carboxyl reactive group through whichthe tri-lysine may be attached to the oligomer. The further conjugate,such as lipophilic/hydrophobic moiety may be attached to the lysineresidue that is attached to the oligomer. In some embodiments, theconjugate (C) is not a monovalent GalNac. The invention also providesLNA antisense oligonucleotides which are conjugated to anasialoglycoprotein receptor targeting moiety. In some embodiments, theconjugate moiety (such as the third region or region C) comprises anasialoglycoprotein receptor targeting moiety, such as galactose,galactosamine, N-formyl-galactosamine, Nacetylgalactosamine,N-propionyl-galactosamine, N-n-butanoyl-galactosamine, andN-isobutanoylgalactos-amine. In some embodiments the conjugate comprisesa galactose cluster, such as N-acetylgalactosamine trimer. In someembodiments, the conjugate moiety comprises a GalNac(N-acetylgalactosamine), such as a mono-valent, di-valent, tri-valent oftetra-valent GalNac. Trivalent GalNac conjugates may be used to targetthe compound to the liver. GalNac conjugates have been used withmethylphosphonate and PNA antisense oligonucleotides (e.g. U.S. Pat. No.5,994,517 and Hangeland et al., Bioconjug Chem. 1995 November-December;6(6):695-701) and siRNAs (e.g. WO2009/126933, WO2012/089352 &WO2012/083046). The GalNac references and the specific conjugates usedtherein are hereby incorporated by reference. WO2012/083046 disclosesGalNac conjugate moieties which comprise cleavable pharmacokineticmodulators, the preferred pharmacokinetic modulators are C16 hydrophobicgroups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. The '046cleavable pharmacokinetic modulators may also be cholesterol. The '046targeting moieties may be selected from the group consisting of:galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine,Npropionyl-galactosamine, N-n-butanoyl-galactosamine,N-iso-butanoylgalactos-amine, galactose cluster, andN-acetylgalactosamine trimer and may have a pharmacokinetic modulatorselected from the group consisting of: hydrophobic group having 16 ormore carbon atoms, hydrophobic group having 16-20 carbon atoms,palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl,dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clustersdisclosed in '046 include: (E)-hexadec-8-enoyl (C16), oleyl (C18),(9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl(C12), C-20 acyl, C24 acyl, dioctanoyl (2×C8). According to '046, thetargeting moiety-pharmacokinetic modulator targeting moiety may belinked to the polynucleotide via a physiologically labile bond or, e.g.a disulfide bond, ora PEG linker.

Other conjugate moieties can include, for example, oligosaccharides andcarbohydrate clusters such as Tyr-Glu-Glu-(aminohexyl GalNAc)₃(YEE(ahGalNAc)₃; a glycotripeptide that binds to Gal/GalNAc receptors onhepatocytes, see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297);lysine-based galactose clusters (e.g., L3G4; Biessen, et al., Cardovasc.Med., 1999, 214); and cholane-based galactose clusters (e.g.,carbohydrate recognition motif for asialoglycoprotein receptor). Furthersuitable conjugates can include oligosaccharides that can bind tocarbohydrate recognition domains (CRD) found on theasiologlycoprotein-receptor (ASGP-R). Example conjugate moietiescontaining oligosaccharides and/or carbohydrate complexes are providedin U.S. Pat. No. 6,525,031, which is incorporated herein by reference inits entirety.

Pharmacokinetic Modulators

The compound of the invention may further comprise one or moreadditional conjugate moieties, of which lipophilic or hydrophobicmoieties are particularly interesting, such as when the conjugate groupis a carbohydrate moiety. Such lipophilic or hydrophobic moieties mayact as pharmacokinetic modulators, and may be covalently linked toeither the carbohydrate conjugate, a linker linking the carbohydrateconjugate to the oligomer or a linker linking multiple carbohydrateconjugates (multi-valent) conjugates, or to the oligomer, optionally viaa linker, such as a bio cleavable linker.

The oligomer or conjugate moiety may therefore comprise apharmacokinetic modulator, such as a lipophilic or hydrophobic moieties.Such moieties are disclosed within the context of siRNA conjugates inWO2012/082046. The hydrophobic moiety may comprise a C8-C36 fatty acid,which may be saturated or un-saturated. In some embodiments, C10, C12,C14, C16, C18, C20, C22, C24, C26, C28, C30, C32 and C34 fatty acids maybe used. The hydrophobic group may have 16 or more carbon atoms.Exemplary suitable hydrophobic groups may be selected from the groupcomprising: sterol, cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. According toWO'346, hydrophobic groups having fewer than 16 carbon atoms are lesseffective in enhancing polynucleotide targeting, but they may be used inmultiple copies (e.g. 2×, such as 2×C8 or C10, C12 or C14) to enhanceefficacy. Pharmacokinetic modulators useful as polynucleotide targetingmoieties may be selected from the group consisting of: cholesterol,alkyl group, alkenyl group, alkynyl group, aryl group, aralkyl group,aralkenyl group, and aralkynyl group, each of which may be linear,branched, or cyclic. Pharmacokinetic modulators are preferablyhydrocarbons, containing only carbon and hydrogen atoms. However,substitutions or heteroatoms which maintain hydrophobicity, for examplefluorine, may be permitted.

Surprisingly, the present inventors have found that GalNac conjugatesfor use with LNA oligomers do not require a pharmacokinetic modulator,and as such, in some embodiments, the GalNac conjugate is not covalentlylinked to a lipophilic or hydrophobic moiety, such as those describedhere in, e.g. do not comprise a C8-C36 fatty acid or a sterol. Theinvention therefore also provides for LNA oligomer GalNac conjugateswhich do not comprise a lipophilic or hydrophobic pharmacokineticmodulator or conjugate moiety/group.

Lipophilic Conjugates

The compounds of the invention may be conjugates comprising of theoligomer (A) and a lipophilic conjugate (C). The biocleavable linker (B)has found to be particularly effective in maintaining or enhancing theactivity of such oligomer conjugates. In some embodiments the conjugategroup (C) and or linker group (Y) comprises a lipophilic group.

Representative conjugate moieties can include lipophilic molecules(aromatic and non-aromatic) including sterol and steroid molecules.Lipophilic conjugate moieties can be used, for example, to counter thehydrophilic nature of an oligomeric compound and enhance cellularpenetration. Lipophilic moieties include, for example, steroids andrelated compounds such as cholesterol (U.S. Pat. No. 4,958,013 andLetsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553),thiocholesterol (Oberhauser et al, Nucl Acids Res., 1992, 20, 533),lanosterol, coprostanol, stigmasterol, ergosterol, calciferol, cholicacid, deoxycholic acid, estrone, estradiol, estratriol, progesterone,stilbestrol, testosterone, androsterone, deoxycorticosterone, cortisone,17-hydroxycorticosterone, their derivatives, and the like.

Other lipophilic conjugate moieties include aliphatic groups, such as,for example, straight chain, branched, and cyclic alkyls, alkenyls, andalkynyls. The aliphatic groups can have, for example, 5 to about 50, 6to about 50, 8 to about 50, or 10 to about 50 carbon atoms. Examplealiphatic groups include undecyl, dodecyl, hexadecyl, heptadecyl,octadecyl, nonadecyl, terpenes, bornyl, adamantyl, derivatives thereofand the like. In some embodiments, one or more carbon atoms in thealiphatic group can be replaced by a heteroatom such as O, S, or N(e.g., geranyloxyhexyl). Further suitable lipophilic conjugate moietiesinclude aliphatic derivatives of glycerols such as alkylglycerols,bis(alkyl)glycerols, tris(alkyl)glycerols, monoglycerides, diglycerides,and triglycerides. In some embodiments, the lipophilic conjugate isdi-hexyldecyl-rac-glycerol or 1,2-di-O-hexyldecyl-rac-glycerol(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea, et al., Nuc.Acids Res., 1990, 18, 3777) or phosphonates thereof. Saturated andunsaturated fatty functionalities, such as, for example, fatty acids,fatty alcohols, fatty esters, and fatty amines, can also serve aslipophilic conjugate moieties. In some embodiments, the fattyfunctionalities can contain from about 6 carbons to about 30 or about 8to about 22 carbons. Example fatty acids include, capric, caprylic,lauric, palmitic, myristic, stearic, oleic, linoleic, linolenic,arachidonic, eicosenoic acids and the like.

In further embodiments, lipophilic conjugate groups can be polycyclicaromatic groups having from 6 to about 50, 10 to about 50, or 14 toabout 40 carbon atoms. Example polycyclic aromatic groups includepyrenes, purines, acridines, xanthenes, fluorenes, phenanthrenes,anthracenes, quinolines, isoquinolines, naphthalenes, derivativesthereof and the like. [0037] Other suitable lipophilic conjugatemoieties include menthols, trityls (e.g., dimethoxytrityl (DMT)),phenoxazines, lipoicacid, phospholipids, ethers, thioethers (e.g.,hexyl-S-tritylthiol), derivatives thereof and the like. Preparation oflipophilic conjugates of oligomeric compounds are well-described in theart, such as in, for example, Saison-Behmoaras et al, EMBOJ., 1991, 10,1111; Kabanov et al., FEBSLett., 1990, 259, 327; Svinarchuk et al,Biochimie, 1993, 75, 49; (Mishra et al., Biochim. Biophys. Acta, 1995,1264, 229, and Manoharan et al., Tetrahedron Lett., 1995, 36, 3651.

Oligomeric compounds containing conjugate moieties with affinity for lowdensity lipoprotein (LDL) can help provide an effective targeteddelivery system. High expression levels of receptors for LDL on tumorcells makes LDL an attractive carrier for selective delivery of drugs tothese cells (Rump, et al., Bioconjugate Chem., 1998, 9, 341; Firestone,Bioconjugate Chem., 1994, 5, 105; Mishra, et al., Biochim. Biophys.Acta, 1995, 1264, 229). Moieties having affinity for LDL include manylipophilic groups such as steroids (e.g., cholesterol), fatty acids,derivatives thereof and combinations thereof. In some embodiments,conjugate moieties having LDL affinity can be dioleyl esters of cholicacids such as chenodeoxycholic acid and lithocholic acid.

In some embodiments, the conjugate group is or may comprise a lipophilicmoiety, such as a sterol (for example, cholesterol, cholesteryl,cholestanol, stigmasterol, cholanic acid and ergosterol). In someembodiments, the conjugate is or may comprise cholesterol. See forexample, Soutschek et al., Nature (2004) 432, 173; Krutzfeldt Nature2005, NAR 2007.

In some embodiments, the conjugate is, or may comprise a lipid, aphospholipid or a lipophilic alcohol, such as a cationic lipids, aneutral lipids, sphingolipids, and fatty acids such as stearic, oleic,elaidic, linoleic, linoleaidic, linolenic, and myristic acids. In someembodiments the fatty acid comprises a C4-C30 saturated or unsaturatedalkyl chain. The alkyl chain may be linear or branched.

In some embodiments, the lipophilic conjugates may be or may comprisebiotin. In some embodiments, the lipophilic conjugate may be or maycomprise a glyceride or glyceride ester.

Lipophilic conjugates, such as cholesterol or as disclosed herein, maybe used to enhance delivery of the oligonucleotide to, for example, theliver (typically hepatocytes).

The following references refer to the use of lipophilic conjugates:Kobylanska et al., Acta Biochim Pol. (1999); 46(3): 679-91. Felber etal., Biomaterials (2012) 33(25): 599-65); Grijalvo et al., J Org Chem(2010) 75(20): 6806-13. Koufaki et al., Curr Med Chem (2009) 16(35):4728-42. Godeau et al J. Med. Chem. (2008) 51(15): 4374-6.

Polymer Conjugates

Conjugate moieties can also include polymers. Polymers can provide addedbulk and various functional groups to affect permeation, cellulartransport, and localization of the conjugated oligomeric compound. Forexample, increased hydrodynamic radius caused by conjugation of anoligomeric compound with a polymer can help prevent entry into thenucleus and encourage localization in the cytoplasm. In someembodiments, the polymer does not substantially reduce cellular uptakeor interfere with hybridization to a complementary strand or othertarget. In further embodiments, the conjugate polymer moiety has, forexample, a molecular weight of less than about 40, less than about 30,or less than about 20 kDa. Additionally, polymer conjugate moieties canbe water-soluble and optionally further comprise other conjugatemoieties such as peptides, carbohydrates, drugs, reporter groups, orfurther conjugate moieties.

In some embodiments, polymer conjugates include polyethylene glycol(PEG) and copolymers and derivatives thereof. Conjugation to PEG hasbeen shown to increase nuclease stability of an oligomeric compound. PEGconjugate moieties can be of any molecular weight including for example,about 100, about 500, about 1000, about 2000, about 5000, about 10,000and higher. In some embodiments, the PEG conjugate moieties contains atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 15, at least 20, or at least 25 ethylene glycolresidues. In further embodiments, the PEG conjugate moiety contains fromabout 4 to about 10, about 4 to about 8, about 5 to about 7, or about 6ethylene glycol residues. The PEG conjugate moiety can also be modifiedsuch that a terminal hydroxyl is replaced by alkoxy, carboxy, acyl,amido, or other functionality. Other conjugate moieties, such asreporter groups including, for example, biotin or fluorescein can alsobe attached to a PEG conjugate moiety. Copolymers of PEG are alsosuitable as conjugate moieties. [0047] Preparation and biologicalactivity of polyethylene glycol conjugates of oligonucleotides aredescribed, for example, in Bonora, et al., Nucleosides Nucleotides,1999, 18, 1723; Bonora, et al., Farmaco, 1998, 53, 634; Efimov, Bioorg.Khim. 1993, 19, 800; and Jaschke, et al, Nucleic Acids Res., 1994, 22,4810. Further example PEG conjugate moieties and preparation ofcorresponding conjugated oligomeric compounds is described in, forexample, U.S. Pat. Nos. 4,904,582 and 5,672,662, each of which isincorporated by reference herein in its entirety. Oligomeric compoundsconjugated to one or more PEG moieties are available commercially.

Other polymers suitable as conjugate moieties include polyamines,polypeptides, polymethacrylates (e.g., hydroxylpropyl methacrylate(HPMA)), poly(L-lactide), poly(DL lactide-co-glycolide (PGLA),polyacrylic acids, polyethylenimines (PEI), polyalkylacrylic acids,polyurethanes, polyacrylamides, N-alkylacrylamides, polyspermine (PSP),polyethers, cyclodextrins, derivatives thereof and co-polymers thereof.Many polymers, such as PEG and polyamines have receptors present incertain cells, thereby facilitating cellular uptake. Polyamines andother amine-containing polymers can exist in protonated form atphysiological pH, effectively countering an anionic backbone of someoligomeric compounds, effectively enhancing cellular permeation. Someexample polyamines include polypeptides (e.g., polylysine,polyornithine, polyhistadine, polyarginine, and copolymers thereof),triethylenetetraamine, spermine, polyspermine, spermidine,synnorspermidine, C-branched spermidine, and derivatives thereof.Preparation and biological activity of polyamine conjugates aredescribed, for example, in Guzaev, et al, Bioorg. Med. Chem. Lett.,1998, 8, 3671; Corey, et al, J Am. Chem. Soc, 1995, 117, 9373; andPrakash, et al, Bioorg. Med. Chem. Lett. 1994, 4, 1733. Examplepolypeptide conjugates of oligonucleotides are provided in, for example,Wei, et al., Nucleic Acids Res., 1996, 24, 655 and Zhu, et al.,Antisense Res. Dev., 1993, 3, 265. Dendrimeric polymers can also be usedas conjugate moieties, such as described in U.S. Pat. No. 5,714,166,which is incorporated herein by reference in its entirety. [0049] Asdiscussed above for polyamines and related polymers, otheramine-containing moieties can also serve as suitable conjugate moietiesdue to, for example, the formation of cationic species at physiologicalconditions. Example amine-containing moieties include 3-aminopropyl,3-(N,N-dimethylamino)propyl, 2-(2-(N,N-dimethylamino)ethoxy)ethyl,2-(N-(2-aminoethyl)-N-methylaminooxyjethyl, 2-(l-imidazolyl)ethyl, andthe like. The G-clamp moiety can also serve as an amine-containingconjugate moiety (Lin, et al., J. Am. Chem. Soc, 1998, 120, 8531).

In some embodiments, the conjugate may be, or may comprise a polymer,such as a polymer selected from the group consisting ofpolyethyleneglycol (PEG), polyamidoamine (PAA), polyethylene oxide andpolyethylenimine (PEI). Galactose, lactose, n-acetylgalactosamine,mannose, mannose-6-phosphate n some embodiments, the polymer is apolycationic polymer. In some embodiments, conjugate moieties can be, orbased on (include) cationic polymers. Numerous studies have demonstratedthat cationic polymers such as cationic albumin can greatly enhancedelivery to particular cell types and/or tissues (e.g. brain delivery,see Lu, W. et. al. (2005) J of Control Release 107:428-448). Given thebenefits of these molecules, the conjugate moieties can be cationicpolymers such as polyethyleneimine, dendrimers, poly(alkylpyridinium)salts, or cationic albumin. In some embodiments is a hydrophilicpolymer. In some embodiments, the polymer is Poly(vinylpyrrolidone)(PVP). In some embodiments, the polymer is a polyamine or polyamide(e.g. U.S. Pat. No. 7,816,337 & U.S. Pat. No. 5,525,465. For polymerconjugates see for example, Zhao et al., Bioconjugate Chem 2005, 16,758-766); Kim et al., J. Control Release (2006) 116; 123. Pettit et al.,Ther. Deliv. (2011) 2(7): 907-17. Yang et al., Bioconjug Chem (2009)20(2): 213-21. Winkler et al (2009) Eur J Med Chem 44(2): 670-7. Zelikinet al, Biomacromolecules (2007) 8(9): 2950-3. See also WO12092373 whichrefers to enzyme cleavable polynucleotide delivery conjugates.

Protein and Peptide Conjugates

Other conjugate moieties can include proteins, subunits, or fragmentsthereof. Proteins include, for example, enzymes, reporter enzymes,antibodies, receptors, and the like. In some embodiments, proteinconjugate moieties can be antibodies or fragments thereof (Kuijpers, etal, Bioconjugate Chem., 1993, 4, 94). Antibodies can be designed to bindto desired targets such as tumor and other disease-related antigens. Infurther embodiments, protein conjugate moieties can be serum proteinssuch as HAS or glycoproteins such as asialoglycoprotein (Rajur, et al.,Bioconjugate Chem., 1997, 6, 935). In yet further embodiments,oligomeric compounds can be conjugated to RNAi-related proteins,RNAi-related protein complexes, subunits, and fragments thereof. Forexample, oligomeric compounds can be conjugated to Dicer or RISC. [0067]Intercalators and minor groove binders (MGBs) can also be suitable asconjugate moieties. In some embodiments, the MGB can contain repeatingDPI (1,2-dihydro-3H-pyrrolo(2,3-e)indole-7-carboxylate) subunits orderivatives thereof (Lukhtanov, et al., Bioconjugate Chem., 1996, 7, 564and Afonina, et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 3199).Suitable intercalators include, for example, polycyclic aromatics suchas naphthalene, perylene, phenanthridine, benzophenanthridine,phenazine, anthraquinone, acridine, and derivatives thereof. Hybridintercalator/ligands include the photonuclease/intercalator ligand6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoyl-pentafluorophenyl ester. This compound is both an acridine moietythat is an intercalator and a p-nitro benzamido group that is aphotonuclease. [0069] In further embodiments, cleaving agents can serveas conjugate moieties. Cleaving agents can facilitate degradation oftarget, such as target nucleic acids, by hydrolytic or redox cleavagemechanisms. Cleaving groups that can be suitable as conjugate moietiesinclude, for example, metallocomplexes, peptides, amines, enzymes, andconstructs containing constituents of the active sites of nucleases suchas imidazole, guanidinium, carboxyl, amino groups, etc.). Examplemetallocomplexes include, for example, Cu-terpyridyl complexes,Fe-porphyrin complexes, Ru-complexes, and lanthanide complexes such asvarious Eu(III) complexes (Hall, et al., Chem. Biol, 1994, 1, 185;Huang, et al., J. Biol. Inorg. Chem., 2000, 5, 85; and Baker, et al,Nucleic Acids Res., 1999, 27, 1547). Other metallocomplexes withcleaving properties include metalloporphyrins and derivatives thereof.Example peptides with target cleaving properties include zinc fingers(U.S. Pat. No. 6,365,379; Lima, et al., Proc. Natl. Acad. Sci. USA,1999, 96, 10010). Example constructs containing nuclease active siteconstituents include bisimiazole and histamine.

Conjugate moieties can also include peptides. Suitable peptides can havefrom 2 to about 30, 2 to about 20, 2 to about 15, or 2 to about 10 aminoacid residues. Amino acid residues can be naturally or non-naturallyoccurring, including both D and L isomers. In some embodiments, peptideconjugate moieties are pH sensitive peptides such as fusogenic peptides.Fusogenic peptides can facilitate endosomal release of agents such asoligomeric compounds to the cytoplasm. It is believed that fusogenicpeptides change conformation in acidic pH, effectively destabilizing theendosomal membrane thereby enhancing cytoplasmic delivery of endosomalcontents. Example fusogenic peptides include peptides derived frompolymyxin B, influenza HA2, GALA, KALA, EALA, melittin-derived peptide,a-helical peptide or Alzheimer beta-amyloid peptide, and the like.Preparation and biological activity of oligonucleotides conjugated tofusogenic peptides are described in, for example, Bongartz, et al.,Nucleic Acids Res., 1994, 22, 4681 and U.S. Pat. Nos. 6,559,279 and6,344,436. Other peptides that can serve as conjugate moieties includedelivery peptides which have the ability to transport relatively large,polar molecules (including peptides, oligonucleotides, and proteins)across cell membranes. Example delivery peptides include Tat peptidefrom HIV Tat protein and Ant peptide from Drosophila antenna protein.Conjugation of Tat and Ant with oligonucleotides is described in, forexample, Astriab-Fisher, et al., Biochem. Pharmacol, 2000, 60, 83. Theseand other delivery peptides that can be used as conjugate moieties areprovided below in Table I:

Conjugated delivery peptides can help control localization of oligomericcompounds to specific regions of a cell, including, for example, thecytoplasm, nucleus, nucleolus, and endoplasmic reticulum (ER). Nuclearlocalization can be effected by conjugation of a nuclear localizationsignal (NLS). In contrast, cytoplasmic localization can be facilitatedby conjugation of a nuclear export signal (NES). [0054] Peptidessuitable for localization of conjugated oligomeric compounds in thenucleus include, for example, N,N-dipalmitylglycyl-apo E peptide orN,N-dipalmitylglycyl-apolipoprotein E peptide (dpGapoE) (Liu, et al,Arterioscler. Thromb. Vase. Biol, 1999, 19, 2207; Chaloin, et al.,Biochem. Biophys. Res. Commun., 1998, 243, 601). Nucleus or nucleolarlocalization can also be facilitated by peptides having arginine and/orlysine rich motifs, such as in HIV-1 Tat, FXR2P, and angiogenin derivedpeptides (Lixin, et al, Biochem. Biophys. Res. Commun., 2001, 284, 185).Additionally, the nuclear localization signal (NLS) peptide derived fromSV40 antigen T (Branden, et al., Nature Biotech, 1999, 17, 784) can beused to deliver conjugated oligomeric compounds to the nucleus of acell. Other suitable peptides with nuclear or nucleolar localizationproperties are described in, for example, Antopolsky, et al.,Bioconjugate Chem., 1999, 10, 598; Zanta, et al., Proc. Natl. Acad. Sci.USA, 1999 (simian virus 40 large tumor antigen); Hum. Mol. Genetics,2000, 9, 1487; and FEBSLett., 2002, 532, 36).

In some embodiments, the delivery peptide for nucleus or nucleolarlocalization comprises at least three consecutive arginine residues orat least four consecutive arginine residues. Nuclear localization canalso be facilitated by peptide conjugates containing RS, RE, or RDrepeat motifs (Cazalla, et al., Mol Cell. Biol, 2002, 22, 6871). In someembodiments, the peptide conjugate contains at least two RS, RE, or RDmotifs.

Localization of oligomeric compounds to the ER can be effected by, forexample, conjugation to the signal peptide KDEL (SEQ ID NO: 18) (Arar,et al., Bioconjugate Chem., 1995, 6, 573; Pichon, et al., Mol.Pharmacol. 1997, 57, 431). [0057] Cytoplasmic localization of oligomericcompounds can be facilitated by conjugation to peptides having, forexample, a nuclear export signal (NES) (Meunier, et al., Nucleic AcidsRes., 1999, 27, 2730). NES peptides include the leucine-rich NESpeptides derived from HIV-1 Rev (Henderson, et al., Exp. Cell Res.,2000, 256, 213), transcription factor III A, MAPKK, PKI-alpha, cyclinB1, and actin (Wada, et al., EMBO J., 1998, 17, 1635) and relatedproteins. Antimicrobial peptides, such as dermaseptin derivatives, canalso facilitate cytoplasmic localization (Hariton-Gazal, et al.,Biochemistry, 2002, 41, 9208). Peptides containing RG and/or KS repeatmotifs can also be suitable for directing oligomeric compounds to thecytoplasm. In some embodiments, the peptide conjugate moieties containat least two RG motifs, at least two KS motifs, or at least one RG andone KS motif. [0058] As used throughout, “peptide” includes not only thespecific molecule or sequence recited herein (if present), but alsoincludes fragments thereof and molecules comprising all or part of therecited sequence, where desired functionality is retained. In someembodiments, peptide fragments contain no fewer than 6 amino acids.Peptides can also contain conservative amino acid substitutions that donot substantially change its functional characteristics. Conservativesubstitution can be made among the following sets of functionallysimilar amino acids: neutral-weakly hydrophobic (A, G, P, S, T),hydrophilic-acid amine (N, D, Q, E), hydrophilic-basic (I, M, L, V), andhydrophobic-aromatic (F, W, Y). Peptides also include homologouspeptides. Homology can be measured according to percent identify using,for example, the BLAST algorithm (default parameters for shortsequences). For example, homologous peptides can have greater than 50,60, 70, 80, 90, 95, or 99 percent identity. Methods for conjugatingpeptides to oligomeric compounds such as oligonucleotides is describedin, for example, U.S. Pat. No. 6,559,279, which is incorporated hereinby reference in its entirety.

In some embodiments, the conjugate moiety is or comprises a protein orpeptide. In some embodiments the peptide is a cell penetrating peptides,e.g. Penetratin, transportan, Peptaibol (e.g. trichorovin-XIIa(TV-XIIa)), TAT peptide (HIV). In some embodiments, the peptide ispolyarginine (e.g. stearyl-(RxR)(4)). In some embodiments the peptide isN-(2-hydroxypropyl) methacrylamide (HPMA) containing tetrapeptideGly-Phe-Leu-Gly (GFLG). In some embodiments, the peptide is abeta-amyloid peptide. In some embodiments the protein or peptide in anantibody or antigen binding site containing fragment thereof (epitopebinding site). In some embodiments the conjugate is or comprisesM6P-HPMA-GFLG (see Yang et al 2009). In some embodiments, the conjugateis or comprises arginine rich peptides (WO2005/115479)—see alsoWO09005793 RGD peptides. In some embodiments, the conjugate is orcomprises a protein carrier (e.g. albumin, albumin-PEGconjugate—RGD-PEG-albumin) (Kang et al) see also WO09045536. In someembodiments, the conjugate is or comprises histidylated oligolysine(e.g. WO0032764). In some embodiments, the conjugate is or comprisesGlycoproteins: transferrin-polycation (e.g. U.S. Pat. No. 5,354,844,WO9217210, WO9213570). In some embodiments, the conjugate is orcomprises asialoglycoprotein (U.S. Pat. No. 5,346,696). In someembodiments, the conjugate is or comprises a polycationic protein (e.g.US603095). In some embodiments, the conjugate is or comprisespoly-pseudo-lysine conjugates (e.g. WO07113531).

Reporter and Dye Conjugate Groups

Reporter groups that are suitable as conjugate moieties include anymoiety that can be detected by, for example, spectroscopic means.Example reporter groups include dyes, flurophores, phosphors,radiolabels, and the like. In some embodiments, the reporter group isbiotin, flourescein, rhodamine, coumarin, or related compounds. Reportergroups can also be attached to other conjugate moieties. In someembodiments, the conjugate is or comprises a label or dye, such as afluorophore, such as FAM (Carboxyfluorescein).

Cross-linking agents can also serve as conjugate moieties. Cross-linkingagents facilitate the covalent linkage of the conjugated oligomericcompounds with other compounds. In some embodiments, cross-linkingagents can covalently link double-stranded nucleic acids, effectivelyincreasing duplex stability and modulating pharmacokinetic properties.In some embodiments, cross-linking agents can be photoactive or redoxactive. Example cross-linking agents include psoralens which canfacilitate interstrand cross-linking of nucleic acids by photoactivation(Lin, et al, Faseb J, 1995, 9, 1371). Other cross-linking agentsinclude, for example, mitomycin C and analogs thereof (Maruenda, et al.,Bioconjugate Chem., 1996, 7, 541; Maruenda, et al., Anti-Cancer DrugDes., 1997, 12, 473; and Huh, et al, Bioconjugate Chem., 1996, 7, 659).Cross-linking mediated by mitomycin C can be effected by reductiveactivation, such as, for example, with biological reductants (e.g.,NADPH-cytochrome c reductase/NADPH system). Further photo-crosslinkingagents include aryl azides such as, for example,N-hydroxysucciniimidyl-4-azidobenzoate (HSAB) andN-succinimidyl-6(-4′-azido-2′-nitrophenyl-amino)hexanoate (SANPAH). Arylazides conjugated to oligonucleotides effect crosslinking with nucleicacids and proteins upon irradiation. They can also crosslink with earnerproteins (such as KLH or BSA).

Various Functional Conjugate Groups

Other suitable conjugate moieties include, for example, polyboranes,carboranes, metallopolyboranes, metallocarborane, derivatives thereofand the like (see, e.g., U.S. Pat. No. 5,272,250, which is incorporatedherein by reference in its entirety).

Many drugs, receptor ligands, toxins, reporter molecules, and othersmall molecules can serve as conjugate moieties. Small moleculeconjugate moieties often have specific interactions with certainreceptors or other biomolecules, thereby allowing targeting ofconjugated oligomeric compounds to specific cells or tissues. Examplesmall molecule conjugate moieties include mycophenolic acid (inhibitorof inosine-5′-monophosphate dihydrogenase; useful for treating psoriasisand other skin disorders), curcumin (has therapeutic applications topsoriasis, cancer, bacterial and viral diseases). In furtherembodiments, small molecule conjugate moieties can be ligands of serumproteins such as human serum albumin (HSA). Numerous ligands of HSA areknown and include, for example, arylpropionic acids, ibuprofen,warfarin, phenylbutazone, suprofen, carprofen, fenfufen, ketoprofen,aspirin, indomethacin, (S)-(+)-pranoprofen, dansylsarcosine,2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid,benzothiadiazide, chlorothiazide, diazepines, indomethicin, barbituates,cephalosporins, sulfa drugs, antibacterials, antibiotics (e.g.,puromycin and pamamycin), and the like. Oligonucleotide-drug conjugatesand their preparation are described in, for example, WO 00/76554, whichis incorporated herein by reference in its entirety.

In some embodiments, the conjugate may be or comprise a small molecule,such as a small molecule drug or pro-drug. Certain drugs are highlyeffective at targeting specific target tissue or cells, and as such theymay be used to target an oligonucleotide to its intended site of action.Furthermore, the small molecule may in itself have a therapeuticactivity, typically once cleaved from the oligonucleotide component ofthe conjugate. Examples include bisphosphonates (widely used for thetreatment of osteoporosis and effective in targeting bone tissues),anti-cancer drugs and chemotherapeutic agents (e.g. doxorubicin ormitomycein C—see U.S. Pat. No. 5,776,907). In some embodiments, the drugmay be a nucleoside analogue, such as a nucleoside polymerase inhibitor.

In yet further embodiments, small molecule conjugates can target or bindcertain receptors or cells. T-cells are known to have exposed aminogroups that can form Schiff base complexes with appropriate molecules.Thus, small molecules containing functional groups such as aldehydesthat can interact or react with exposed amino groups can also besuitable conjugate moieties. Tucaresol and related compounds can beconjugated to oligomeric compounds in such a way as to leave thealdehyde free to interact with T-cell targets. Interaction of tucaresolwith T-cells in believed to result in therapeutic potentiation of theimmune system by Schiff-base formation (Rhodes, et al., Nature, 1995,377, 6544).

In some embodiments, the conjugate is or comprises a (e.g. cell surface)receptor ligand. In some embodiments the conjugate is or comprises afolate receptor ligand, such as a folic acid group—see for example,EP1572067 or WO2005/069994, WO2010/045584). Other cell surface receptorligands include antibodies and fragments thereof, prostate-specificmembrane antigen, neuron surface antigens (see WO2011/131693)

In some embodiments, the conjugate moieties are ligands for receptors orcan associate with molecules that (in turn) associate with receptors.Included in this class are bile acids, small molecule drug ligands,vitamins, aptamers, carbohydrates, peptides (including but not limitedto hormones, proteins, protein fragments, antibodies or antibodyfragments), viral proteins (e.g. capsids), toxins (e.g. bacterialtoxins), and more. Also included in this class are conjugates that aresteroidal in nature e.g. cholesterol, cholestanol, cholanic acid,stigmasterols, pregnolones, progesterones, corticosterones,aldosterones, testosterones, estradiols, ergosterols, and more),Preferred conjugate moieties of the disclosure are cholesterol (CHOL),cholestanol (CHLN), cholanic acid (CHLA), stigmasterol (STIG), andergosterol (ERGO). In certain preferred embodiments, the conjugatemoiety is cholesterol.

In some embodiments the conjugate comprises a sterol, such ascholesterol or tocopherol, optionally including a linker, such as afatty acid linker, e.g. a C6 linker. In some embodiments the conjugatescomprise Conj5a or Conj 6a.

Conjugate moieties can also include vitamins. Vitamins are known to betransported into cells by numerous cellular transport systems.Typically, vitamins can be classified as water soluble or lipid soluble.Water soluble vitamins include thiamine, riboflavin, nicotinic acid orniacin, the vitamin B6 pyridoxal group, pantothenic acid, biotin, folicacid, the B]2 cobamide coenzymes, inositol, choline and ascorbic acid.Lipid soluble vitamins include the vitamin A family, vitamin D, thevitamin E tocopherol family and vitamin K (and phytols). Relatedcompounds include retinoid derivatives such as tazarotene andetretinate. [0040] In some embodiments, the conjugate moiety includesfolic acid folate) and/or one or more of its various forms, such asdihydrofolic acid, tetrahydrofolic acid, folinic acid, pteropolyglutamicacid, dihydrofolates, tetrahydrofolates, tetrahydropterins, 1-deaza,3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-dideaza, 5,10-dideaza,8,10-dideaza and 5,8-dideaza folate analogs, and antifolates. Folate isinvolved in the biosynthesis of nucleic acids and therefore impacts thesurvival and proliferation of cells. Folate cofactors play a role in theone-carbon transfers that are needed for the biosynthesis of pyrimidinenucleosides. Cells therefore have a system of transporting folates intothe cytoplasm. Folate receptors also tend to be overexpressed in manyhuman cancer cells, and folate-mediated targeting of oligonucleotides toovarian cancer cells has been reported (Li, et al, Pharm. Res. 1998, 15,1540, which is incorporated herein by reference in its entirety).Preparation of folic acid conjugates of nucleic acids are described in,for example, U.S. Pat. No. 6,528,631, which is incorporated herein byreference in its entirety.

Vitamin conjugate moieties include, for example, vitamin A (retinol)and/or related compounds. The vitamin A family (retinoids), includingretinoic acid and retinol, are typically absorbed and transported totarget tissues through their interaction with specific proteins such ascytosol retinol-binding protein type II (CRBP-II), retinol-bindingprotein (RBP), and cellular retinol-binding protein (CRBP). The vitaminA family of compounds can be attached to oligomeric compounds via acidor alcohol functionalities found in the various family members. Forexample, conjugation of an N-hydroxy succinimide ester of an acid moietyof retinoic acid to an amine function on a linker pendant to anoligonucleotide can result in linkage of vitamin A compound to theoligomeric compound via an amide bond. Also, retinol can be converted toits phosphoramidite, which is useful for 5′ conjugation.alpha-Tocopherol (vitamin E) and the other tocopherols (beta throughzeta) can be conjugated to oligomeric compounds to enhance uptakebecause of their lipophilic character. Also, vitamin D, and itsergosterol precursors, can be conjugated to oligomeric compounds throughtheir hydroxyl groups by first activating the hydroxyl groups to, forexample, hemisuccinate esters. Conjugation can then be effected directlyto the oligomeric compound or to an arninolinker pendant from theoligomeric compound. Other vitamins that can be conjugated to oligomericcompounds in a similar manner on include thiamine, riboflavin,pyridoxine, pyridoxamine, pyridoxal, deoxypyridoxine. Lipid solublevitamin K's and related quinone-containing compounds can be conjugatedvia carbonyl groups on the quinone ring. The phytol moiety of vitamin Kcan also serve to enhance binding of the oligomeric compounds to cells.

Other functional groups which may be used as conjugates in compounds ofthe invention, include imidazole conjugate—RNase A catalytic centermimics (polyamine-imidazole conjugates)—see Guerniou et al Nucleic AcidsRes (2007); 35 (20): 6778-87.

Conjugates are typically non-nucleotide moieties. However, in thecontext of blocking groups or targeting groups, or nucleotide analogsmall therapeutics, it is recognized that the oligonucleotide may becovalently linked to a nucleotide moiety via the DNA/RNA phosphodiesterregion of the invention. Suitably, a nucleic acid group, as used in thecontext of the invention may, in some embodiments, lack complementarityto the target of the oligonucleotide (region A).

In some embodiments, the blocking or targeting moiety is an aptamer (seee.g. Meng et al., PLoS One (2012) 7(4): e33434, WO2005/111238 &WO12078637).

A blocking group may also be or comprise a oligonucleotide region whichis complementary to, e.g. part of, the antisense oligonucleotide. Inthis regard the blocking oligonucleotide is covalently bound to anantisense oligonucleotide via the DNA/RNA phosphodiester region (regionb), and optionally a linker. The blocking oligonucleotide is, in someembodiments, therefore able to form a duplex with the antisenseoligonucleotide. Suitably the blocking nucleotide sequence (as thirdregion or region C) is a short oligonucleotide sequence of e.g. 3-10nucleotides in length which forms a duplex (i.e. is complementary to)with an equivalent length of the first region. In some embodiments alinker is used between the second region and the blocking region.

Like delivery peptides, nucleic acids can also serve as conjugate likemoieties that can affect localization of conjugated oligomeric compoundsin a cell. For example, nucleic acid conjugate moieties can contain polyA, a motif recognized by poly A binding protein (PABP), which canlocalize poly A-containing molecules in the cytoplasm (Gorlach, et al.,Exp. Cell Res., 1994, 211, 400. In some embodiments, the nucleic acidconjugate moiety contains at least 3, at least 4, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 15, atleast 20, and at least 25 consecutive A bases. The nucleic acidconjugate moiety can also contain one or more AU-rich sequence elements(AREs). AREs are recognized by ELAV family proteins which can facilitatelocalization to the cytoplasm (Bollig, et al, Biochem. Bioophys. Res.Commun., 2003, 301, 665). Example AREs include UUAUUUAUU and sequencescontaining multiple repeats of this motif. In other embodiments, thenucleic acid conjugate moiety contains two or more AU or AUU motifs.Similarly, the nucleic acid conjugate moiety can also contain one ormore CU-rich sequence elements (CREs) (Wein, et al, Eur. J. Biochem.,2003, 270, 350) which can bind to proteins HuD and/or HuR of the ELAVfamily of proteins. As with AREs, CREs can help localize conjugatedoligomeric compounds to the cytoplasm. In some embodiments, the nucleicacid conjugate moiety contains the motif (CUUU)n, wherein, for example,n can be 1 to about 20, 1 to about 15, or 1 to about 11. The (CUUU)nmotif can optionally be followed or preceded by one or more U. In someembodiments, n is about 9 to about 12 or about 11. The nucleic acidconjugate moiety can also include substrates of hnRNP proteins(heterogeneous nuclear ribonucleoprotein), some of which are involved inshuttling nucleic acids between the nucleus and cytoplasm, (e.g., nhRNPAl and nhRNP K; see, e.g., Mili, et al, Mol. Cell Biol, 2001, 21, 7307).Some example hnRNP substrates include nucleic acids containing thesequence UAGGA/U or (GG)ACUAGC(A). Other nucleic acid conjugate moietiescan include Y strings or other tracts that can bind to, for example,HnRNP I. In some embodiments, the nucleic acid conjugate can contain atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20, and at least 25consecutive pyrimidine bases. In other embodiments the nucleic acidconjugate can contain greater than 50, greater than 60, greater than 70,greater than 80, greater than 90, or greater than 95 percent pyrimidinebases.

Other nucleic acid conjugate-like moieties can include pumilio (pufprotein) recognition sequences such as described in Wang, et al., Cell,2002, 110, 501. Example pumilio recognition sequences can includeUGUANAUR, where N can be any base and R can be a purine base.Localization to the cytoplasm can be facilitated by nucleic acidconjugate moieties containing AREs and/or CREs. Nucleic acidconjugate-like moieties serving as substrates of hnRNPs can facilitatelocalization of conjugated oligomeric compounds to the cytoplasm (e.g.,hnRNP Al or K) or nucleus (e.g., hnRNP I). Additionally, nucleuslocalization can be facilitated by nucleic acid conjugate-like moietiescontaining polypyrimidine tracts.

A Reactive Group

A reactive group is a group which is used in chemical synthesis, whichin the context of the present invention may be used “conjugate” theoligonucleotide, or otherwise covalently link the oligonucleotide to thethird region (X), such as the conjugate, blocking group or targetinggroup, or optionally the linker (Y). An example of a reactive group is aphosphoramidite, which is widely used in oligonucleotide synthesis.

An Activation Group

An activation group is a group which may be activated to form a reactivegroup. In this respect, an activation group may be considered as aprotected reactive group, which may be deprotected prior to enable useof the reactive group, for example in the methods ofsynthesis/manufacture disclosed herein.

Linkage Group

A nucleoside linkage is the linkage group either between nucleosides inthe oligonucleotide, or, when present, may also describe the group whichattaches the third region (X or C) or the linker (Y) to region B—forexample this linker may be a phosphate (containing) linkage group or atriazol group.

Blocker Group (Also Referred to as a Blocking/Blocker Moiety)

In some aspects, the third region is a blocking region. A blocker istypically a conjugate or an oligonucleotide (typically not complementaryto the target region), which, for example (but not limited to) eitherthrough steric hindrance, or through hybridization to the first region(or first and second regions), prevents or reduces activity of theoligomer. The (blocked) activity may be against its intended target (thetarget) or in some embodiments unintended targets (off-targets).

The oligomeric compound of the invention may therefore comprise a firstregion, such as a gapmer or LNA gaper oligonucleotide (such as a gapmerof formula X′Y′Z), a second region which is a biocleavable linker, suchas region B as described herein, and a third region, region C, whichcomprises a region of at least 2 consecutive nucleosides, such as 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides which arecomplementary to a corresponding part of the first region. In someembodiments at least 2 nucleosides of region C, such as 3, 4, 5, 6, 7,8, 9, or 10 nucleosides are high affinity nucleoside analogues, such asLNA (BNA)—in some embodiments, these may form the distal part of regionC. The high affinity nucleoside analogues of region C may form acontiguous sequence of high affinity nucleoside analogues, which may beflanked by other nucleosides, such as DNA nucleosides (also part ofregion C, referred to as the proximal part of region C). In someembodiments, region C comprises between 2-8 (such as 3, 4, 5, 6, & 7 LNA(BNA) nucleotides, and in the same or in a different embodiment a regionof between 2-16 DNA nucleotides (such as 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15). In some embodiments, the distal part of region Bcomprises a contiguous region of high affinity nucleotide analogues, forexample a contiguous region of 2, 3, 4, 5, 6, 7, or 8 LNA nucleotides.The proximal region may comprise a contiguous region of non-LNAnucleotides, such as those referred to herein, such as DNA nucleotides,such as a region of 2-16 non-LNA nucleotides. It is however alsounderstood that the proximal region may comprise high affinitynucleotide analogues including LNA, but as contiguous regions of LNA canrestrict the conformational flexibility of the proximal region (which isthought to act as a loop) it may, in some embodiments be useful to limitthe use of long stretches of LNA in the proximal (or loop forming part),such as no more than 4 consecutive LNAs, such as no more than 3consecutive LNAs, or no more than 2 consecutive LNAs.

In some embodiments, the region of other nucleotides in region C (suchas DNA nucleotides) forms a contiguous sequence with region B, i.e. isproximal to the terminal nucleotide of region B), so that the region ofhigh affinity nucleotides is distal to region B. In such an embodiment,region B and the proximal part of region C (e.g. the region comprisingDNA nucleotides) may form a flexible loop, which allows the distal partof region C to hybridize with the first region. The proximal part ofregion C may or may not be complementary to a corresponding part ofregion A. In some embodiments, the distal part of region C iscomplementary to nucleotides which form a region which is capable ofrecruiting RNaseH, such as the gap region of a gapmer (referred toherein region Y′). In such an embodiment, the blocking region (region C)forms a duplex with the gap region, or part thereof, thereby blockingthe availability of the central region of the gapmer to interact withother molecules or the target or off-targets. The invention thereforeprovides a solution to the inherent toxicity of DNA phosphorothioateoligonucleotides (which are typically used for the gap region ofgapmers), as it allows for the controlled activation of gapmer oligomers(region A) within the target tissue or cells. In this respect, the useof a blocking region can act as a pro-drug. It is recognized that theblocking region (region C or distal part thereof), may also be directedtowards other regions of an oligomer, including a mixmer ortotalmeroligomer, or the flanking regions of a gapmer, or across the wing regionand the gap region of a gapmer. In such an embodiment, the hybridizationor region C (or distal part thereof) to region A (or part of region A),prevents the hybridization of the corresponding part of region A tobiomolecules, and may therefore also be used to prevent unintendedinteraction with other biomolecules, enhancing specificity, tissuespecific activity, and diminishing the risk of toxicity. Theinternucleoside linkages between the nucleotides of region C may beother than phosphodiester, such as may be phosphorothioate.

Targeting Group (Also Referred to as a Targeting Moiety)

A targeting moiety is a group whose presence on the oligomeric compoundcauses a differential pattern of biodistribution and/or cellular uptakeof the oligomeric compound. Targeting groups may be, for example,receptor ligands, antibodies, hormones or hormone analogues, aptamersetc. The examples show the use of cholesterol as a targetinggroup—cholesterol is recognized by the LDL receptor in the surface ofhepatocytes, resulting in the preferential uptake of cholesterolconjugated oligonucleotides into the liver. The examples also illustratethe use of GalNac, tocopherol, and folic acid as targeting groups.

Oligomer Linked Biocleavable Conjugates

The oligomeric compound may optionally, comprise a second region (regionB) which is positioned between the oligomer (referred to as region A)and the conjugate (referred to as region C). Region B may be a linkersuch as a cleavable linker (also referred to as a physiologically labilelinkage).

In some embodiments, the compound of the invention comprises abiocleavable linker (also referred to as the physiologically labilelinker, Nuclease Susceptible Physiological Labile Linkages, or nucleasesusceptible linker), for example the phosphate nucleotide linker (suchas region B) or a peptide linker, which joins the oligomer (orcontiguous nucleotide sequence or region A), to a conjugate moiety (orregion C).

Biocleavable linkers according to the present invention refers tolinkers which are susceptible to cleavage in a target tissue (i.e.physiologically labile), for example liver and/or kidney. It ispreferred that the cleavage rate seen in the target tissue is greaterthan that found in blood serum. Suitable methods for determining thelevel (%) of cleavage in tissue (e.g. liver or kidney) and in serum arefound in example 6. In some embodiments, the biocleavable linker (alsoreferred to as the physiologically labile linker, or nucleasesusceptible linker), such as region B, in a compound of the invention,are at least about 20% cleaved, such as at least about 30% cleaved, suchas at least about 40% cleaved, such as at least about 50% cleaved, suchas at least about 60% cleaved, such as at least about 70% cleaved, suchas at least about 75% cleaved, in the the liver or kidney homogenateassay of Example 6. In some embodiments, the cleavage (%) in serum, asused in the assay in Example 6, is less than about 20%, such as lessthan about 10%, such as less than 5%, such as less than about 1%.

Biocleavable linkers according to the present invention refers tolinkers which are susceptible to cleavage in a target tissue (i.e.physiologically labile), for example liver and/or kidney. It ispreferred that the cleavage rate seen in the target tissue is greaterthan that found in blood serum. Suitable methods for determining thelevel (%) of cleavage in tissue (e.g. liver or kidney) and in serum arefound in example 6. In some embodiments, the biocleavable linker (alsoreferred to as the physiologically labile linker, or nucleasesusceptible linker), such as region B, in a compound of the invention,are at least about 20% cleaved, such as at least about 30% cleaved, suchas at least about 40% cleaved, such as at least about 50% cleaved, suchas at least about 60% cleaved, such as at least about 70% cleaved, suchas at least about 75% cleaved, in the liver or kidney homogenate assayof Example 6. In some embodiments, the cleavage (%) in serum, as used inthe assay in Example 6, is less than about 30%, is less than about 20%,such as less than about 10%, such as less than 5%, such as less thanabout 1%.

In some embodiments, which may be the same of different, thebiocleavable linker (also referred to as the physiologically labilelinker, or nuclease susceptible linker), such as region B, in a compoundof the invention, are susceptible to S1 nuclease cleavage.Susceptibility to S1 cleavage may be evaluated using the S1 nucleaseassay shown in Example 6. In some embodiments, the biocleavable linker(also referred to as the physiologically labile linker, or nucleasesusceptible linker), such as region B, in a compound of the invention,are at least about 30% cleaved, such as at least about 40% cleaved, suchas at least about 50% cleaved, such as at least about 60% cleaved, suchas at least about 70% cleaved, such as at least about 80% cleaved, suchas at least about 90% cleaved, such as at least 95% cleaved after 120min incubation with S1 nuclease according to the assay used in Example6.

Nuclease Susceptible Physiological Labile Linkages: In some embodiments,the oligomer (also referred to as oligomeric compound) of the invention(or conjugate) comprises three regions:

-   -   i) a first region (region A), which comprises 10-18 contiguous        nucleotides;    -   ii) a second region (region B) which comprises a biocleavable        linker    -   iii) a third region (C) which comprises a conjugate moiety, a        targeting moiety, an activation moiety, wherein the third region        is covalent linked to the second region.

In some embodiments, region B may be a phosphate nucleotide linker. Forexample such linkers may be used when the conjugate is a lipophilicconjugate, such as a lipid, a fatty acid, sterol, such as cholesterol ortocopherol. Phosphate nucleotide linkers may also be used for otherconjugates, for example carbohydrate conjugates, such as GalNac.

Peptide and Other Linkers

In some embodiments, the biocleavable linker (region B) is a peptide,such as a trilysine peptide linker which may be used in a polyGalNacconjugate, such a triGalNac conjugate. Other linkers known in the artwhich may be used, including disulfide linkers (also referred to asdithio or disulphide herein). Other peptide linkers include, e.g. aTyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide.

Phosphate Nucleotide Linkers

In some embodiments, region B comprises between 1-6 nucleotides, whichis covalently linked to the 5′ or 3′ nucleotide of the first region,such as via a internucleoside linkage group such as a phosphodiesterlinkage, wherein either

-   -   a. the internucleoside linkage between the first and second        region is a phosphodiester linkage and the nucleoside of the        second region [such as immediately] adjacent to the first region        is either DNA or RNA; and/or    -   b. at least 1 nucleoside of the second region is a        phosphodiester linked DNA or RNA nucleoside;

In some embodiments, region A and region B form a single contiguousnucleotide sequence of 10-22, such as 12-20 nucleotides in length.

In some aspects the internucleoside linkage between the first and secondregions may be considered part of the second region.

Linkers

A linkage or linker is a connection between two atoms that links onechemical group or segment of interest to another chemical group orsegment of interest via one or more covalent bonds. Conjugate moieties(or targeting or blocking moieties) can be attached to the oligomericcompound directly or through a linking moiety (linker or tether)—alinker. Linkers are bifunctional moieties that serve to covalentlyconnect a third region, e.g. a conjugate moiety, to an oligomericcompound (such as to region B). In some embodiments, the linkercomprises a chain structure or an oligomer of repeating units such asethylene glyol or amino acid units. The linker can have at least twofunctionalities, one for attaching to the oligomeric compound and theother for attaching to the conjugate moiety. Example linkerfunctionalities can be electrophilic for reacting with nucleophilicgroups on the oligomer or conjugate moiety, or nucleophilic for reactingwith electrophilic groups. In some embodiments, linker functionalitiesinclude amino, hydroxyl, carboxylic acid, thiol, phosphoramidate,phosphate, phosphite, unsaturations (e.g., double or triple bonds), andthe like. Some example linkers include 8-amino-3,6-dioxaoctanoic acid(ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC), 6-aminohexanoic acid (AHEXor AHA), 6-aminohexyloxy,4-aminobutyric acid, 4-aminocyclohexylcarboxylic acid, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate) (LCSMCC),succinimidyl m-maleimido-benzoylate (MBS), succinimidylN-e-maleimido-caproylate (EMCS), succinimidyl6-(beta-maleimido-propionamido) hexanoate (SMPH), succinimidylN-(a-maleimido acetate) (AMAS), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB), beta-alanine (beta-ALA),phenylglycine (PHG), 4-aminocyclohexanoic acid (ACHC),beta-(cyclopropyl) alanine (beta-CYPR), amino dodecanoic acid (ADC),alylene diols, polyethylene glycols, amino acids, and the like.

A wide variety of further linker groups are known in the art that can beuseful in the attachment of conjugate moieties to oligomeric compounds.A review of many of the useful linker groups can be found in, forexample, Antisense Research and Applications, S. T. Crooke and B.Lebleu, Eds., CRC Press, Boca Raton, Fla., 1993, p. 303-350. A disulfidelinkage has been used to link the 3′ terminus of an oligonucleotide to apeptide (Corey, et al., Science 1987, 238, 1401; Zuckermann, et al, JAm. Chem. Soc. 1988, 110, 1614; and Corey, et al., J Am. Chem. Soc.1989, 111, 8524). Nelson, et al., Nuc. Acids Res. 1989, 17, 7187describe a linking reagent for attaching biotin to the 3′-terminus of anoligonucleotide. This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol iscommercially available from Clontech Laboratories (Palo Alto, Calif.)under the name 3′-Amine. It is also commercially available under thename 3′-Amino-Modifier reagent from Glen Research Corporation (Sterling,Va.). This reagent was also utilized to link a peptide to anoligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991,32, 879. A similar commercial reagent for linking to the 5′-terminus ofan oligonucleotide is 5′-Amino-Modifier C6. These reagents are availablefrom Glen Research Corporation (Sterling, Va.). These compounds orsimilar ones were utilized by Krieg, et al, Antisense Research andDevelopment 1991, 1, 161 to link fluorescein to the 5′-terminus of anoligonucleotide. Other compounds such as acridine have been attached tothe 3′-terminal phosphate group of an oligonucleotide via apolymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA1984, 81, 3297). [0074]Any of the above groups can be used as a singlelinker or in combination with one or more further linkers.

Linkers and their use in preparation of conjugates of oligomericcompounds are provided throughout the art such as in WO 96/11205 and WO98/52614 and U.S. Pat. Nos. 4,948,882; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025;5,254,469; 5,245,022; 5,112,963; 5,391,723; 5,510475; 5,512,667;5,574,142; 5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437,each of which is incorporated by reference in its entirety.

As used herein, a physiologically labile bond is a labile bond that iscleavable under conditions normally encountered or analogous to thoseencountered within a mammalian body (also referred to as a cleavablelinker). Physiologically labile linkage groups are selected such thatthey undergo a chemical transformation (e.g., cleavage) when present incertain physiological conditions. Mammalian intracellular conditionsinclude chemical conditions such as pH, temperature, oxidative orreductive conditions or agents, and salt concentration found in oranalogous to those encountered in mammalian cells. Mammalianintracellular conditions also include the presence of enzymatic activitynormally present in a mammalian cell such as from proteolytic orhydrolytic enzymes. In some embodiments, the cleavable linker issusceptible to nuclease(s) which may for example, be expressed in thetarget cell—and as such, as detailed herein, the linker may be a shortregion (e.g. 1-10) phosphodiester linked nucleosides, such as DNAnucleosides,

Chemical transformation (cleavage of the labile bond) may be initiatedby the addition of a pharmaceutically acceptable agent to the cell ormay occur spontaneously when a molecule containing the labile bondreaches an appropriate intra- and/or extra-cellular environment. Forexample, a pH labile bond may be cleaved when the molecule enters anacidified endosome. Thus, a pH labile bond may be considered to be anendosomal cleavable bond. Enzyme cleavable bonds may be cleaved whenexposed to enzymes such as those present in an endosome or lysosome orin the cytoplasm. A disulfide bond may be cleaved when the moleculeenters the more reducing environment of the cell cytoplasm. Thus, adisulfide may be considered to be a cytoplasmic cleavable bond. As usedherein, a pH-labile bond is a labile bond that is selectively brokenunder acidic conditions (pH<7). Such bonds may also be termedendosomally labile bonds, since cell endosomes and lysosomes have a pHless than 7.

Activated Oligomers

In some embodiments, the invention provides an activated oligomer—i.e.an intermediate used in the synthesis of the oligomer of theinvention—e.g. the conjugated oligomer. In this respect, the oligomer ofthe invention may, in some embodiments comprise region A and region B asdescribed herein, and region B in covalently linked to an activation (orreactive) group, suitable for use in conjugation of the oligomer.

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected, e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl,ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl ortrifluoroacetyl. In some embodiments, thefunctional moiety is self-cleaving. In other embodiments, the functionalmoiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which isincorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis. In some embodiments, theoligomers are functionalized with a hindered ester containing anaminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_(W),wherein w is an integer ranging from 1 to 10, preferably about 6,wherein the alkyl portion of the alkylamino group can be straight chainor branched chain, and wherein the functional group is attached to theoligomer via an ester group (—O—C(O)—(CH₂)_(W)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH)

In some embodiments, sulfhydryl-activated oligonucleotides areconjugated with polymer moieties such as polyethylene glycol or peptides(via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular by methodsdisclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(0-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more monomers is prepared using areagent such as, for example,5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention may haveamine-containing functional moieties on the nucleobase, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Methods of Synthesis and Manufacture

The invention also provides methods of synthesis or manufacture of theoligomer of the invention. The oligomer may be made using standardoligonucleotide synthesis, which is typically performed on a solidsupport, such as a universal support. As illustrated in FIGS. 5-10, theoligomer of the invention may be synthesized, for example, by thesequential synthesis of the first region and the second region, followedby the addition (e.g. conjugation) of the third region (X) optionallyvia a linker (Y). Region Y, when present may be joined to the region B,and region X subsequently added to region Y, or region Y and X may beadded to region B in a single reaction step.

Alternatively, the oligomer synthesis my occur via the initial couplingof region X, or region X and Y to the oligonucleotide support column,followed by sequential oligonucleotide synthesis of region B and thenregion A.

Alternatively, the use of a cleavable bidirectional group attached tothe oligonucleotide synthesis support (in an initial or pre-step),allows for a method where the oligonucleotide regions B and A aresynthesized on one reactive group of the bifunctional group, and regionX or region X and Y are synthesized on a second reactive group of thebifunctional group, wherein the oligonucleotide synthesis or addition ofX (or X and Y) to the support may occur in any order or even together.The cleavage of the bifunctional group from the support then producesthe oligomer of the invention. The bifunctional group may for example bea nucleoside, where one entity (e.g. region B or X or X—Y—) is attachedto a phosphate containing group on the nucleoside (e.g. a 5′ or 3′group), and the other (e.g. region B or X or X—Y—), is attached, forexample to an reactive group present on the nucleobase.

Alternatively region X or X—Y may be joined to the oligomer (region B)after oligonucleotide synthesis, such as after the cleavage step. Theinvention therefore also relates to the intermediate oligomer, whichcomprises regions A and B, and a reactive or activation group attachedto region B, which is subsequently used to join region X or regions Xand Y to region B.

Region Y or region X may be linked to region B as a phosphoramidite, forexample—allowing for the formation of the oligomer in a singleoligonucleotide synthesis, followed by cleavage of the oligomer from theoligonucleotide synthesis support (US). In this regard, in someembodiments, the linkage group between region B and region X or Y may bea phosphate containing group, such as a nucleoside linkage, such asphosphodiester, phosphorothioate, phosphorodithioate, boranophosphate,methylphosphonate or others, such as those referred to herein.Alternatively other chemical linkages may be used such as a triazolgroup.

In some embodiments, the third region (X) or X—Y— may be linked toregion B via a group other than a 5′ or 3′ phosphate, for example via areactive group at another position, for example a reactive group, suchas an amine on the base of a nucleoside in region B.

Oligonucleotide synthesis may occur in the 5′-3′ direction, or, as istypical of most oligonucleotide synthesis, in the 3′-5′ direction.

In some non-limiting examples, the oligonucleotide-conjugate constructcan be assembled in different ways, e.g.

-   -   A) The B-A part of the construct can be made on an        oligonucleotide synthesis machine capable of synthesizing both        phosphorothioate and phosphorodiester linkages. B-A can then        optionally be elongated by standard phosphoramidite chemistry        using a building block X-A-P (conjugate moiety with linker        attached) to create X-A-B-A or with building block X-P        (conjugate moiety with no linker) to create X-B-A

-   -   B) The B-A part of the construct can be made on an        oligonucleotide synthesis machine capable of synthesizing both        phosphorthioate and phosphordiester linkages. B-A can then        optionally be sequentially elongated by standard phosphoramidite        chemistry using a building block DMTrO-A-P followed by building        block X-P to create X-A-B-A with a PO or PS linkage between the        X and A part.

The B-A part of the construct can be made on an oligonucleotidesynthesis machine capable of synthesizing both phosphorthioate andphosphordiester linkages. B-A can then optionally be sequentiallyelongated by standard phosphoramidite chemistry using a building blockPGN-A-P to create H₂N-A-B-A. After cleavage and deprotection of theoligonucleotide the free amine of the oligonucleotide can be conjugatedwith moiety X in which a functional group of X has been activated inorder to react with the terminal primary amine of the oligonucleotide.

Compositions

The oligomer of the invention may be used in pharmaceutical formulationsand compositions. Suitably, such compositions comprise apharmaceutically acceptable diluent, carrier, salt or adjuvant.WO2007/031091 provides suitable and preferred pharmaceuticallyacceptable diluent, carrier and adjuvants—which are hereby incorporatedby reference. Suitable dosages, formulations, administration routes,compositions, dosage forms, combinations with other therapeutic agents,pro-drug formulations are also provided in WO2007/031091—which are alsohereby incorporated by reference.

Antisense oligonucleotides may be admixed with pharmaceuticallyacceptable active or inert substances for the preparation ofpharmaceutical compositions or formulations. Compositions and methodsfor the formulation of pharmaceutical compositions are dependent upon anumber of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

An antisense compound can be utilized in pharmaceutical compositions bycombining the antisense compound with a suitable pharmaceuticallyacceptable diluent or carrier. A pharmaceutically acceptable diluentincludes phosphate-buffered saline (PBS). PBS is a diluent suitable foruse in compositions to be delivered parenterally.

Pharmaceutical compositions comprising antisense compounds encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other oligonucleotide which, upon administration to an animal,including a human, is capable of providing (directly or indirectly) thebiologically active metabolite or residue thereof.

Accordingly, for example, the disclosure is also drawn topharmaceutically acceptable salts of antisense compounds, prodrugs,pharmaceutically acceptable salts of such prodrugs, and otherbioequivalents. Suitable pharmaceutically acceptable salts include, butare not limited to, sodium and potassium salts. A prodrug can includethe incorporation of additional nucleosides at one or both ends of anantisense compound which are cleaved by endogenous nucleases within thebody, to form the active antisense compound. In this regard the prodrugmay comprise region B and a conjugate, targeting or blocking moiety asaccording to the present invention. In some embodiments, the oligomer ofthe invention is a pro-drug.

The use of lipophilic conjugates according to the invention allows forthe incorporation of the oligomer of the invention into lipidoids orliposomes, e.g. cationic liposomes (e.g. cationic liposome SNALPs(stable nucleic acid lipid particle), which are particularly useful fordelivery of oligomers e.g. to the liver, e.g. siRNAs.

Applications

The oligomers of the invention may be utilized as research reagents for,for example, diagnostics, therapeutics and prophylaxis.

In research, in some embodiments, such oligomers may be used tospecifically inhibit the synthesis of protein (typically by degrading orinhibiting the mRNA and thereby prevent protein formation) in cells andexperimental animals thereby facilitating functional analysis of thetarget or an appraisal of its usefulness as a target for therapeuticintervention.

For therapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of thetarget is treated by administering oligomeric compounds in accordancewith this invention. Further provided are methods of treating a mammal,such as treating a human, suspected of having or being prone to adisease or condition, associated with expression of the target byadministering a therapeutically or prophylactically effective amount ofone or more of the oligomers or compositions of the invention. Theoligomer, a conjugate or a pharmaceutical composition according to theinvention is typically administered in an effective amount.

The invention also provides for the use of the compound or conjugate ofthe invention as described for the manufacture of a medicament for thetreatment of a disorder as referred to herein, or for a method of thetreatment of as a disorder as referred to herein.

The invention also provides for a method for treating a disorder asreferred to herein said method comprising administering a compoundaccording to the invention as herein described, and/or a conjugateaccording to the invention, and/or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

Medical Indications

In some embodiments, the disease is cancer. In some embodiments, thedisease is an inflammatory disease. In some embodiments, the disease isa cardiovascular disease, such as

In some embodiments the disease or disorder is myocardial infarction(MI).

In some embodiments, the disease or disorder is, or results in or isassociated with fibrosis, such as liver-fibrosis, cardiac fibrosis orlocal fibrosis.

In some embodiments, the disease or disorder is blood clotting disorder.

In some embodiments the disease or disorder is or comprises (results inor is associated with) bone-lose.

In some embodiments, the disease or disorder is a liver disease ordisorder.

In some embodiments the disease or disorder is a metabolic disorder,which may for example be a liver disease or disorder, and/or in someaspects a cardiovascular disease or disorder).

Cardiovascular/Metabolic diseases include, for examples, metabolicsyndrome, obesity, hyperlipidemia, HDL/LDL cholesterol imbalance,dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquiredhyperlipidemia, statin-resistant, hypercholesterolemia, coronary arterydisease (CAD), and coronary heart disease (CHD)., atherosclerosis, heartdisease, diabetes (I and/or II), NASH, acute coronary syndrome (ACS),NASH, chronic heart failure, cardiovascular disease, cardio metabolicdisease, hyperlipidaemia and related disorders, metabolic syndrome,atherosclerosis, chronic heart failure, vascular disease, peripheralarterial disease, heart disease, ischemia, type 2 diabetes, type 1diabetes,

In some embodiments, the disease or disorder is selected from the groupconsisting of metabolic syndrome, obesity, hyperlipidemia,atherosclerosis, HDL/LDL cholesterol imbalance, dyslipidemias, e.g.,familial combined hyperlipidemia (FCHL), acquired hyperlipidemia,statin-resistant, hypercholesterolemia, coronary artery disease (CAD),and coronary heart disease (CHD).

In some embodiments, the disease or disorder is selected from the groupconsisting of chronic heart failure, cardiovascular disease, cardiometabolic disease, chronic heart failure, vascular disease, peripheralarterial disease, heart disease, ischemia, acute coronary syndrome(ACS).

In some embodiments, the disease or disorder is type 2 diabetes, type 1diabetes,

In some embodiments, the disease or disorder is a viral disease, such aspolycythemia, hepatitis C, hepatitis B, BKV, HIV.

In some embodiments, the disease or disorder is a severe and rarediseases (or genetic disorder).

The invention further provides use of a compound of the invention in themanufacture of a medicament for the treatment of a disease, disorder orcondition, such as those as referred to herein.

Generally stated, some aspects of the invention is directed to a methodof treating a mammal suffering from or susceptible to conditionsassociated with abnormal levels of the target, comprising administeringto the mammal and therapeutically effective amount of an oligomertargeted to the target that comprises one or more LNA units. Theoligomer, a conjugate or a pharmaceutical composition according to theinvention is typically administered in an effective amount.

An interesting aspect of the invention is directed to the use of thecompound as defined herein for the preparation of a medicament for thetreatment of a disease, disorder or condition as referred to herein.

Moreover, the invention relates to a method of treating a subjectsuffering from a disease or condition such as those referred to herein.

A patient who is in need of treatment is a patient suffering from orlikely to suffer from the disease or disorder.

In some embodiments, the term ‘treatment’ as used herein refers to bothtreatment of an existing disease (e.g. a disease or disorder as hereinreferred to), or prevention of a disease, i.e. prophylaxis. It willtherefore be recognized that treatment as referred to herein may, insome embodiments, be prophylactic.

EXAMPLES

Oligonucleotide List

In the following list, Capital letters represent LNA nucleosides, suchas beta-D-oxy LNA, lower case letters represent DNA nucleosides. CapitalL is a LNA, such as beta-D-oxy, and lower case d is a DNA nucleoside.LNA cytosines are optionally 5′methyl cytosine. The internucleosideswithin region A are phosphorothioate, and within region B arephosphodiester (as shown). The internucleoside linkage between region Aand B is phoshodiester, but where region B is >1 DNA nucleotide, mayoptionally be other than phosphodiester (e.g. may be phosphorothioate).There is, optionally a further linker (Y), between region B and regionC, such as a C6 linker. # refers to SEQ ID No.

ApoB Targeting Compounds

Cleavable Seq (5′-3′) Linker Region C - # (Region A) (Region B)Conjugate 1 GCattggtatTCA no no 2 GCattggtatTCA no Cholesterol 3GCattggtatTCA SS Cholesterol 4 GCattggtatTCA 3PO-DNA (5′tca3′)Cholesterol 5 GCattggtatTCA 2PO-DNA (5′ca3′) Cholesterol 6 GCattggtatTCA1PO-DNA (5′a3′) Cholesterol

PCSK9—Mouse Specific Compounds

Cleavable Seq (5′-3′) Linker Conjugate # (A) (B) (C)  7 GTctgtggaaGCG nono  8 GTctgtggaaGCG no Cholesterol  9 GTctgtggaaGCG 2PO-DNA (5′ca3′)Cholesterol 10 GTctgtggaaGCG 2PO-DNA (5′ct3′) Cholesterol

FVII (Mouse FVII)

Cleavable linker Conjugate # Seq (5′-3′) (B) (C) 11 LLddddddddLLL no no12 LLddddddddLLL GaINAc cluster 13 LLddddddddLLL 2PO (ca) GaINAc cluster14 LLddddddddLLL SS Cholesterol 15 LLddddddddLLL 2PO (ca) Cholesterol

ApoB Targeting Compounds with FAM label conjugates

Cleavable linker Conjugate # Seq (5′-3′) (B) (C) 16 GCattggtatTCA3PO-DNA (5′tca3′) FAM 17 GCattggtatTCA 2PO-DNA (5′ca3′) FAM 18GCattggtatTCA 1PO-DNA (5′a3′) FAM 19 GCattggtatTCA 3PO-DNA (5′gac3′) FAM20 GCattggtatTCA no FAM

Target X compounds (A human therapeutic target)

Cleavable Linker Region C # Seq (5′-3′) (B) (in 3′-end) 21LLLdddddddddLLL 3PO-DNA  5′-dddddLLLL-3′ (5′tgc3′) 22 LLLdddddddddLLL5′-ddddddddLLLL-3′ 23 LLLdddddddddLLL 3PO-DNA  5′-dddddLLLL-3′ (5′tgc3′)24 LLLdddddddddLLL 5′-ddddddddLLLL-3′

In the above compounds, region C comprises the complement to the Seq(Region A) so that the 3′ nucleotide of region C aligns (forms a basepair) with the 8^(th) nucleotide of region A from the 5′ end. Region Ctherefore loops back and forms an 8 base hybridization with region Aacross the 3′ wing of the gapmer and 5 bases of the DNA gap region,thereby creating a “pro-drug” which is inactive until the linker region(B) is cleaved.

APOB Targeting Compounds

Cleavable Linker # Seq-(5′-3′) (B) Conjugate 25 GCattggtatTCA noFolic acid 26 GCattggtatTCA SS Folic acid 27 GCattggtatTCA2PO-DNA (5′ca3′) Folic acid 28 GCattggtatTCA no monoGaINAc 29GCattggtatTCA SS monoGaINAc 30 GCattggtatTCA 2PO-DNA (5′ca3′) monoGaINAc31 GCattggtatTCA no FAM 32 GCattggtatTCA SS FAM 33 GCattggtatTCA2PO-DNA (5′ca3′) FAM 34 GCattggtatTCA no Tocopherol 35 GCattggtatTCA SSTocopherol 36 GCattggtatTCA 2PO-DNA (5′ca3′) Tocopherol

PCSK9 Compounds

# Seq (5′-3′) linker Conjugate 37 TGCtacaaaacCCA no 38 AATgctacaaaaCCCAno 39 AATgctacaaaacCCA no 40 GCtgtgtgagcttGG no 41 TGctgtgtgagctTGG no42 TGCtgtgtgagctTGG no 43 TCCtggtctgtgtTCC no 44 TCCtggtctgtgttCC no 45TGCtacaaaacCCA 2PO-DNA (5′ca3′) Cholesterol 46 AATgctacaaaaCCCA2PO-DNA (5′ca3′) Cholesterol 47 AATgctacaaaacCCA 2PO-DNA (5′ca3′)Cholesterol 48 GCtgtgtgagcttGG 2PO-DNA (5′ca3′) Cholesterol 49TGctgtgtgagctTGG 2PO-DNA (5′ca3′) Cholesterol 50 TGCtgtgtgagctTGG2PO-DNA (5′ca3′) Cholesterol 51 TCCtggtctgtgtTCC 2PO-DNA (5′ca3′)Cholesterol 52 TCCtggtctgtgttCC 2PO-DNA (5′ca3′) Cholesterol

Monkey Study Compounds ApoB PGP-52 DNA

53 GTtgacactgTC No no  5 GCattggtatTCA 2PO-DNA (5′ca3′) Cholesterol 54GTtgacactgTC 2PO-DNA (5′ca3′) Cholesterol 46 AATgctacaaaaCCCA2PO-DNA (5′ca3′) Cholesterol 49 TGctgtgtgagctTGG 2PO-DNA (5′ca3′)Cholesterol

SEQ ID NO 53 is provided as the parent compound of SEQ ID NO 54.

Mouse Experiments: Unless otherwise specified, the mouse experiments maybe performed as follows:

Dose Administration and Sampling:

7-10 week old C57Bl6-N mice were used, animals were age and sex matched(females for study 1, 2 and 4, males in study 3). Compounds wereinjected i.v. into the tail vein. For intermediate serum sampling, 2-3drops of blood were collected by puncture of the vena facialis, finalbleeds were taken from the vena cava inferior. Serum was collected ingel-containing serum-separation tubes (Greiner) and kept frozen untilanalysis.

C57BL6 mice were dosed i.v. with a single dose of 1 mg/kg ASO (or amountshown) formulated in saline or saline alone according to the informationshown. Animals were sacrificed at e.g. day 4 or 7 (or time shown) afterdosing and liver and kidney were sampled. RNA isolation and mRNAanalysis: mRNA analysis from tissue was performed using the QantigenemRNA quantification kit (“bDNA-assay”, Panomics/Affimetrix), followingthe manufacturers protocol. For tissue lysates, 50-80 mg of tissue waslysed by sonication in 1 ml lysis-buffer containing Proteinase K.Lysates were used directly for bDNA-assay without RNA extraction.Probesets for the target and GAPDH were obtained custom designed fromPanomics. For analysis, luminescence units obtained for target geneswere normalized to the housekeeper GAPDH.

Serum analysis for ALT, AST and cholesterol was performed on the “CobasINTEGRA 400 plus” clinical chemistry platform (Roche Diagnostics), using10 μl of serum.

For quantification of Factor VII serum levels, the BIOPHEN FVII enzymeactivity kit (#221304, Hyphen BioMed) was used according to themanufacturer's protocol.

For oligonucleotide quantification, a fluorescently-labeled PNA probe ishybridized to the oligo of interest in the tissue lysate. The samelysates are used as for bDNA-assays, just with exactly weighted amountsof tissue. The heteroduplex is quantified using AEX-HPLC and fluorescentdetection.

Example 1: Synthesis of Compounds SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3,SEQ ID NO 4 and SEQ ID NO 5

Oligonucleotides were synthesized on uridine universal supports usingthe phosphoramidite approach on an Expedite 8900/MOSS synthesizer(Multiple Oligonucleotide Synthesis System) or equivalent at 4 μmolscale. At the end of the synthesis, the oligonucleotides were cleavedfrom the solid support using aqueous ammonia for 1-2 hours at roomtemperature, and further deprotected for 16 hours at 65° C. Theoligonucleotides were purified by reverse phase HPLC (RP-HPLC) andcharacterized by UPLC, and the molecular mass was further confirmed byESI-MS. See below for more details.

Elongation of the Oligonucleotide

The coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu),DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-TorC6-S-S linker) is performed by using a solution of 0.1 M of the5′-O-DMT-protected amidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. For thefinal cycle a commercially available C₆-linked cholesterolphosphoramidite was used at 0.1M in DCM. Thiolation for introduction ofphosphorthioate linkages is carried out by using xanthane hydride (0.01M in acetonitrile/pyridine 9:1). Phosphordiester linkages are introducedusing 0.02 M iodine in THF/Pyridine/water 7:2:1. The rest of thereagents are the ones typically used for oligonucleotide synthesis.

Purification by RP-HPLC;

The crude compounds were purified by preparative RP-HPLC on a PhenomenexJupiter C18 10μ 150×10 mm column. 0.1 M ammonium acetate pH 8 andacetonitrile was used as buffers at a flowrate of 5 mL/min. Thecollected fractions were lyophilized to give the purified compoundtypically as a white solid.

Abbreviations;

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

Example 2: Design of LNA Antisense Oligonucleotides

Oligomers used in the examples and figures. The SEQ # is an identifierused throughout the examples and figures

TABLE 2 SEQ ID NO Compound Sequence Comment  #1 5′-G _(s) ^(o) ^(m) C_(s) ^(o) a_(s)t_(s)t_(s)g_(s)g_(s)a_(s)t_(s)   Mother  T _(s) ^(o) ^(m)C _(s) ^(o) A ^(o)-3′ compound  without conjugated  Cholesterol  #25′-CHOL G _(s) ^(o) ^(m) C _(s) ^(o) a_(s) t_(s) t_(s)   Chol-3833g_(s) g_(s) t_(s) a_(s) t_(s) T _(s) ^(o m) C _(s) ^(o)  A ^(o)-3′  #35′-Chol_C6 C6SSC6 G _(s) ^(o) ^(m) C _(s) ^(o)   Chol-a_(s) t_(s) t_(s) g_(s) g_(s) t_(s) a_(s) t_(s) SS-3833 T _(s) ^(o) ^(m)C _(s) ^(o) A ^(o)-3′  #4 5′-Chol_C6 t c a G _(s) ^(o) ^(m) C _(s)^(o)   Chol- a_(s) t_(s) t_(s) g_(s) g_(s) t_(s) a_(s) t_(s) 3PO-3833 T_(s) ^(o m) C _(s) ^(o) A ^(o)-3′  #5 5′-Chol_C6 c a G _(s) ^(o m) C_(s) ^(o)   Chol- a_(s) t_(s) t_(s) g_(s) g_(s) t_(s) a_(s) t_(s)2PO-3833 T _(s) ^(o m) C _(s) ^(o) A^(o)-3′  #6 5′-Chol_C6 a G_(s)^(o m)C_(s) ^(o) a_(s)   Chol-t_(s) t_(s) g_(s) g_(s) t_(s) a_(s) t_(s) T_(s) ^(o) 1PO-3833 ^(m)C_(s)^(o) A^(o)-3′  #7 5′-G_(s)o T_(s)o c_(s) t_(s) g_(s) t_(s)   Mother g_(s) g_(s) a_(s) a_(s) G_(s)o ^(m)C_(s)o compound  Go-3′ withoutconjugate  #8 5′-Chol_C6 G_(s) ^(o) T_(s) ^(o) c_(s) t_(s)   Chol-4061g_(s) t_(s) g_(s) g_(s) a_(s) a_(s) G_(s) ^(o)  ^(m)C_(s) ^(o) G^(o)-3′ #9 5′-Chol_C6 c a G_(s) ^(o) T_(s) ^(o)   Chol-c_(s) t_(s) g_(s) t_(s) g_(s) g_(s) a_(s) a_(s) 2PO(ca)-4061 G_(s)^(o m)C_(s) ^(o) G^(o)-3′ #10 5′-Chol_C6 c t G_(s) ^(o) T_(s) ^(o)  Chol- c_(s) t_(s) g_(s) t_(s) g_(s) g_(s) a_(s) a_(s) 2PO(ct)-4061 G_(s)^(o m)C_(s) ^(o) G^(o)-3′

Example 3. Knock Down of ApoB mRNA with Cholesterol-Conjugates In Vivo

C57BL6/J mice were injected with a single dose saline or 1 mg/kgunconjugated LNA-antisense oligonucleotide (SEQ ID #1) or equimolaramounts of LNA antisense oligonucleotides conjugated to Cholesterol withdifferent linkers and sacrificed at days 1-10 according to Tab. 3.

RNA was isolated from liver and kidney and subjected to qPCR with ApoBspecific primers and probe to analyze for ApoB mRNA knockdown.

Conclusions: Cholesterol conjugated to an ApoB LNA antisenseoligonucleotide with a linker composed of 2 or 3 DNA withPhophodiester-backbone (Seq #4 and 5) showed a preference for liverspecific knock down of ApoB (FIG. 11). This means increases efficiencyand duration of ApoB mRNA knock down in liver tissue compared to theunconjugated compound (Seq #1), as well as compared to Cholesterolconjugates with stable linker (Seq #2) and with disulphide linker (Seq.#3) and concomitant less knock down activity of Seq #4 and #5 in kidneytissue.

Materials and Methods:

Experimental Design;

TABLE 3 Animal Com- Conc. at Ani- No of strain/ pound dose Gr. mal ani-gender/ Dose level vol. 10 Body Sacri- no. ID no. mals feed per dayml/kg weight fice A 1  1-4  4 C57BL/6J- NaCl — Day- Day ♀-Chow 0.9% 1, 710 and 10 2  5-8  4 C57BL/6J- SEQ ID 0.1 Day- Day ♀-Chow NO 1 mg/ml 1, 710 1 mg/kg and 10 3  9-12 4 C57BL/6J- SEQ ID 0.12 Day- Day ♀-Chow NO 2mg/ml 1, 7 10 1.2 mg/kg and 10 4 13-16 4 C57BL/6J- SEQ ID 0.12 Day- Day♀-Chow NO 3 mg/ml 1, 7 10 1.2 mg/kg and 10 5 17-20 4 C57BL/6J- SEQ ID0.13 Day- Day ♀-Chow NO 4 mg/ml 1, 7 10 1.3 mg/kg and 10 6 21-24 4C57BL/6J- SEQ ID 0.13 Day- Day ♀-Chow NO 5 mg/ml 1, 7 10 1.3 mg/kg and10 B 7 25-28 4 C57BL/6J- NaCl — Day- Day ♀- Chow 0.9% 1, 7 7 8 29-32 4C57BL/6J- SEQ ID 0.1 Day- Day ♀-Chow NO 1 mg/ml 1, 7 7 1 mg/kg 9 33-36 4C57BL/6J- SEQ ID 0.12 Day- Day ♀-Chow NO 2 mg/ml 1, 7 7 1.2 mg/kg 1037-40 4 C57BL/6J- SEQ ID 0.12 Day- Day ♀-Chow NO 3 mg/ml 1, 7 7 1.2mg/kg 11 41-44 4 C57BL/6J- SEQ ID 0.13 Day- Day ♀-Chow NO 4 mg/ml 1, 7 71.3 mg/kg 12 45-48 4 C57BL/6J- SEQ ID 0.13 Day- Day ♀-Chow NO 5 mg/ml 1,7 7 1.3mg/kg C 13 49-52 4 C57BL/6J- NaCl — Day Day ♀-Chow 0.9% 0, 3 3 1453-56 4 C57BL/6J- SEQ ID 0.1 Day Day ♀-Chow NO 1 mg/ml 0, 3 3 1 mg/kg 1557-60 4 C57BL/6J- SEQ ID 0.12 Day Day ♀-Chow NO 2 mg/ml 0, 3 3 1.2 mg/kg16 61-64 4 C57BL/6J- SEQ ID 0.12 Day Day ♀-Chow NO 3 mg/ml 0, 3 3 1.2mg/kg 17 65-68 4 C57BL/6J- SEQ ID 0.13 Day Day ♀-Chow NO 4 mg/ml 0, 3 31.3 mg/kg 18 69-72 4 C57BL/6J- SEQ ID 0.13 Day Day ♀-Chow NO 5 mg/ml 0,3 3 1.3 mg/kg D 19 73-76 4 C57BL/6J- NaCl — Day- Day ♀-Chow 0.9% 1, 1 120 77-80 4 C57BL/6J- SEQ ID 0.1 Day- Day ♀-Chow NO 1 mg/ml 1, 1 1 1mg/kg 21 81-84 4 C57BL/6J- SEQ ID 0.12 Day- Day ♀-Chow NO 2 mg/ml 1, 1 11.2 mg/kg 22 85-88 4 C57BL/6J- SEQ ID 0.12 Day- Day ♀-Chow NO 3 mg/ml 1,1 1 1.2 mg/kg 23 89-92 4 C57BL/6J- SEQ ID 0.13 Day- Day ♀-Chow NO 4mg/ml 1, 1 1 1.3 mg/kg 24 93-96 4 C57BL/6J- SEQ ID 0.13 Day- Day ♀-ChowNO 5 mg/ml 1, 1 1 1.3 mg/kg

Dose administration. C57BL/6JBom female animals, app. 20 g at arrival,were dosed with 10 ml per kg BW (according to day 0 bodyweight) i.v. ofthe compound formulated in saline or saline alone according to table 3.

Sampling of liver and Kidney tissue. The animals were anaesthetized with70% CO₂-30% O₂ and sacrificed by cervical dislocation according to Table3. One half of the large liver lobe and one kidney were minced andsubmerged in RNAIater.

Total RNA Isolation and First strand synthesis. Total RNA was extractedfrom maximum 30 mg of tissue homogenized by bead-milling in the presenceof RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen cat. no. 74106)according to the manufacturer's instructions.

First strand synthesis was performed using Reverse Transcriptasereagents from Ambion according to the manufacturer's instructions.

For each sample 0.5 μg total RNA was adjusted to (10.8 μl) with RNasefree H₂O and mixed with 2 μl random decamers (50 μM) and 4 μl dNTP mix(2.5 mM each dNTP) and heated to 70° C. for 3 min after which thesamples were rapidly cooled on ice. 2 μl 10× Buffer RT, 1 μl MMLVReverse Transcriptase (100 U/μl) and 0.25 μl RNase inhibitor (10 U/μl)were added to each sample, followed by incubation at 42° C. for 60 min,heat inactivation of the enzyme at 95° C. for 10 min and then the samplewas cooled to 4° C. cDNA samples were diluted 1:5 and subjected toRT-QPCR using Taqman Fast Universal PCR Master Mix 2× (AppliedBiosystems Cat #4364103) and Taqman gene expression assay (mApoB,Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers protocoland processed in an Applied Biosystems RT-qPCR instrument (7500/7900 orViiA7) in fast mode.

Example 4. Knock Down of ApoB mRNA with Cholesterol-Conjugates In Vivoand LNA Distribution to Liver and Kidney

C57BL6/J mice were injected with a single dose saline or 1 mg/kgunconjugated LNA-antisense oligonucleotide (SEQ ID #1) or equimolaramounts of LNA antisense oligonucleotides conjugated to Cholesterol withdifferent linkers and sacrificed at days 1-16 according to Tab. 4. RNAwas isolated from liver and kidney and subjected to qPCR with ApoBspecific primers and probe to analyze for ApoB mRNA knockdown.

The LNA oligonucleotide content was measured in liver and kidney usingLNA based sandwich ELISA method.

Conclusions: Cholesterol conjugated to an ApoB LNA antisenseoligonucleotide with a linker composed of 1, 2 or 3 DNA withPhophodiester-backbone (Seq #4, #5 and #6) showed a preference for liverspecific knock down of ApoB (FIG. 14). This means increased efficiencyand duration of ApoB mRNA knock down in liver tissue compared to theunconjugated compound (Seq #1), and concomitant less knock down activityof Seq #4, #5 and #6 in kidney tissue. The Cholesterol conjugated LNAantisense oligonucleotides have a much higher uptake in the liver andmuch lover uptake in the kidney as compared to the unconjugated LNAoligonucleotide (FIG. 15).

Materials and Methods:

Experimental Design;

TABLE 4 Ani- No. of Animal Compound Conc. at Body Sacri- Group mal Ani-strain/ Dose level dose vol. Adm. Dosing weight fice Part no. id no.mals gender/feed per day 10 ml/kg Route day day day A 1 1-3 3 C57BL/Saline — iv 0 0, 1 1 6J/♀/Chow 2 4-6 3 C57BL/ SEQ ID 0.1 iv 0 0, 1 16J/♀/Chow NO 1 mg/ml 1 mg/kg 3 7-9 3 C57BL/ SEQ ID 0.135 iv 0 0, 1 16J/♀/Chow NO 4 mg/ml equimolar 1.35mg/kg 4 10-12 3 C57BL/ SEQ ID 0.128iv 0 0, 1 1 6J/♀/Chow NO 5 mg/ml equimolar 1.28 mg/kg 5 13-15 3 C57BL/SEQ ID 0.121 iv 0 0, 1 1 6J/♀/Chow NO 6 mg/ml equimolar 1.21 mg/kg B 616-18 3 C57BL/ Saline — iv 0 0, 3 3 6J/♀/Chow 7 19-21 3 C57BL/ SEQ ID0.1 iv 0 0, 3 3 6J/♀/Chow NO 1 mg/ml 1 mg/kg 8 22-24 3 C57BL/ SEQ ID0.135 iv 0 0, 3 3 6J/♀/Chow NO 4 mg/ml equimolar 1.35mg/kg 9 25-27 3C57BL/ SEQ ID 0.128 iv 0 0, 3 3 6J/♀/Chow NO 5 mg/ml equimolar 1.28mg/kg 10 28-30 3 C57BL/ SEQ ID 0.121 iv 0 0, 3 3 6J/♀/Chow NO 6 mg/mlequimolar 1.21 mg/kg C 11 31-33 3 C57BL/ Saline — iv 0 0, 3 3 6J/♀/Chow12 34-36 3 C57BL/ SEQ ID 0.1 iv 0 0, 7 7 6J/♀/Chow NO 1 mg/ml 1 mg/kg 1337-39 3 C57BL/ SEQ ID 0.135 iv 0 0, 7 7 6J/♀/Chow NO 4 mg/ml equimolar1.35 mg/kg 14 40-42 3 C57BL/ SEQ ID 0.128 iv 0 0, 7 7 6J/♀/Chow NO 5mg/ml equimolar 1.28 mg/kg 15 43-45 3 C57BL/ SEQ ID 0.121 iv 0 0, 7 76J/♀/Chow NO 6 mg/ml equimolar 1.21 mg/kg D 16 46-48 3 C57BL/ Saline —iv 0 0,7, 10 6J/♀/Chow 10 17 49-51 3 C57BL/ SEQ ID 0.1 iv 0 0,7, 106J/♀/Chow NO 1 mg/ml 10 1 mg/kg 18 52-54 3 C57BL/ SEQ ID 0.135 iv 0 0,7,10 6J/♀/Chow NO 4 mg/ml 10 equimolar 1.35 mg/kg 19 55-57 3 C57BL/ SEQ ID0.128 iv 0 0,7, 10 6J/♀/Chow NO 5 mg/ml 10 equimolar 1.28 mg/kg 20 58-603 C57BL/ SEQ ID 0.121 iv 0 0,7, 10 6J/♀/Chow NO 6 mg/ml 10 equimolar1.21 mg/kg E 21 61-63 3 C57BL/ Saline — iv 0 0,7, 13 6J/♀/Chow 13 2264-66 3 C57BL/ SEQ ID 0.1 iv 0 0,7, 13 6J/♀/Chow NO 1 mg/ml 13 1 mg/kg23 67-69 3 C57BL/ SEQ ID 0.135 iv 0 0,7, 13 6J/♀/Chow NO 4 mg/ml 13equimolar 1.35 mg/kg 24 70-72 3 C57BL/ SEQ ID 0.128 iv 0 0,7, 136J/♀/Chow NO 5 mg/ml 13 equimolar 1.28 mg/kg 25 73-75 3 C57BL/ SEQ ID0.121 iv 0 0,7, 13 6J/♀/Chow NO 6 mg/ml 13 equimolar 1.21 mg/kg F 2676-78 3 C57BL/ Saline — iv 0 0,7, 16 6J/♀/Chow 14,16 27 79-81 3 C57BL/SEQ ID 0.1 iv 0 0,7, 16 6J/♀/Chow NO 1 mg/ml 14,16 1 mg/kg 28 82-84 3C57BL/ SEQ ID 0.135 iv 0 0,7, 16 6J/♀/Chow NO 4 mg/ml 14,16 equimolar1.35 mg/kg 29 85-87 3 C57BL/ SEQ ID 0.128 iv 0 0,7, 16 6J/♀/Chow NO 5mg/ml 14,16 equimolar 1.28 mg/kg 30 88-90 3 C57BL/ SEQ ID 0.121 iv 00,7, 16 6J/♀/Chow NO 6 mg/ml 14,16 equimolar 1.21 mg/kg

Dose administration. C57BL/6JBom female animals, app. 20 g at arrival,were dosed with 10 ml per kg BW (according to day 0 bodyweight) i.v. ofthe compound formulated in saline or saline alone according to Table 4.

Sampling of liver and Kidney tissue. The animals were anaesthetized with70% CO₂-30% O₂ and sacrificed by cervical dislocation according to Table4. One half of the large liver lobe and one kidney were minced andsubmerged in RNAIater.

Total RNA Isolation and First strand synthesis. Total RNA was extractedfrom maximum 30 mg of tissue homogenized by bead-milling in the presenceof RLT-Lysis buffer using the Qiagen RNeasy kit (Qiagen cat. no. 74106)according to the manufacturer's instructions.

First strand synthesis was performed using Reverse Transcriptasereagents from Ambion according to the manufacturer's instructions.

For each sample 0.5 μg total RNA was adjusted to (10.8 μl) with RNasefree H₂O and mixed with 2 μl random decamers (50 μM) and 4 μl dNTP mix(2.5 mM each dNTP) and heated to 70° C. for 3 min after which thesamples were rapidly cooled on ice. 2 μl 10× Buffer RT, 1 μl MMLVReverse Transcriptase (100 U/μl) and 0.25 μl RNase inhibitor (10 U/μl)were added to each sample, followed by incubation at 42° C. for 60 min,heat inactivation of the enzyme at 95° C. for 10 min and then the samplewas cooled to 4° C. cDNA samples were diluted 1:5 and subjected toRT-QPCR using Taqman Fast Universal PCR Master Mix 2× (AppliedBiosystems Cat #4364103) and Taqman gene expression assay (mApoB,Mn01545150_m1 and mGAPDH #4352339E) following the manufacturers protocoland processed in an Applied Biosystems RT-qPCR instrument (7500/7900 orViiA7) in fast mode.

Oligo content sandwich ELISA; Liver and kidney samples (100 mg) werecollected in tubes at different times after dosing. The samples wereadded buffer, (pH 8.0 100 mM NaCl, 25 mM EDTA, 0.25 mM Tris), protease k(1%, Sigma P4850-5) and 2 Tungsten Carbide Beads (3 mm) (Qiagen) andhomogenized for 8 min (Retsch MM300, 25 Hz [l/s]) and incubated thehomogenate at 37° C. over night. The samples are spun at 14000 g for 15minutes before use.

Standards 1-100 μg/g of LNA oligonucleotides in kidney and liver wereprepared and treated as above. Standards and samples were diluted to(100-5000 ng/L) into 150 μl of a 35 nM solution of a biotinylated anddigoxigenin modified capture and detection probe (5×SSCT buffer [(750 mMNaCl, and 75 mM sodium citrate, containing 0.05% (v/v) Tween-20 pH 7.0)]and mixed for an hour. Streptavidin-coated (Nunc ImmobilizerStreptavidin F96 CLEAR module plate Nunc Cat. No. 436014) were washedthree times (5×SSCT buffer, 300 μL). The samples 100 μL was transferredto the streptavidin coated plates and incubated for one hour undergentle shaking. The wells were aspirated and washed three times with 300μl of 2×SSCT buffer (300 mM NaCl+30 mM sodium citrate containing 0.05%(v/v) Tween-20, pH 7.0). One hundred microliters of anti-Dig-AP Fabfragments (Roche Applied Science, Cat. No. 11 093 274 910) diluted1:4000 in PBST (Phosphate buffered saline, pH 7.2) were added to thewells and incubated for 1 hour at room temperature under gentleagitation. The wells were aspirated and washed three times with 300 μlof 2×SSCT buffer. One hundred microliters of substrate solution (KPLBluePhos Microwell Phosphatase substrate system 50-88-00) were added toeach well. The intensity of the color development was measuredspectrophotometrically at 615 nm every 5 minutes after shaking. The testsamples were referenced against the standard samples.

Example 5: Knock Down of PCSK9 mRNA with Cholesterol Conjugates In Vivo

NMRI mice were injected with a single dose saline or 10 mg/kgunconjugated LNA-antisense oligonucleotide (SEQ ID 7) or equimolaramounts of LNA antisense oligonucleotides conjugated to Cholesterol withdifferent linkers and sacrificed at days 1-10 according to Tab. 5.

RNA was isolated from liver and kidney and subjected to qPCR with PCSK9specific primers and probe to analyze for PCSK9 mRNA knockdown.

Conclusions: Cholesterol conjugated to an PCSK9 LNA antisenseoligonucleotide with a linker composed of 2 DNA withPhophodiester-backbone (Seq #9 and #10) showed an enhanced liver knockdown of PCSK9 (FIG. 16) compared to the unconjugated compound (Seq#7),as well as compared to Cholesterol conjugates with stable linker(Seq #8).

Materials and Methods:

Experimental Design;

TABLE 5 Ani- No. of Animal Compound Conc. at Body Sacri- Group mal Ani-strain/ Dose level dose vol. Adm. Dosing weight fice Part no. id no.mals gender/feed per day 10 ml/kg Route day day day A 1 1-3 3 NMRI/♀/Saline — iv 0 0, 1 1 Chow 2 4-6 3 NMRI/♀/ SEQ ID 1 mg/ml iv 0 0, 1 1Chow NO 7 10 mg/kg 3 7-9 3 NMRI/♀/ SEQ ID 1.13 iv 0 0, 1 1 Chow NO 8mg/ml equimolar 11.3 mg/kg 5 13-15 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 1 1Chow NO 9 mg/ml equimolar 12.7 mg/kg 6 16-18 3 NMRI/♀/ SEQ ID 1.27 iv 00, 1 1 Chow NO 10 mg/ml equimolar 12.7 mg/kg B 7 19-21 3 NMRI/♀/ Saline— iv 0 0, 3 3 Chow 8 22-24 3 NMRI/♀/ SEQ ID 1 mg/ml iv 0 0, 3 3 Chow NO7 10 mg/kg 9 25-27 3 NMRI/♀/ SEQ ID 1.13 iv 0 0, 3 3 Chow NO 8 mg/mlequimolar 11.3 mg/kg 11 31-33 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 3 3 Chow NO9 mg/ml equimolar 12.7 mg/kg 12 34-36 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 3 3Chow NO 10 mg/ml equimolar 12.7 mg/kg C 13 37-39 3 NMRI/♀/ Saline — iv 00, 7 7 Chow 14 40-42 3 NMRI/♀/ SEQ ID 1 mg/ml iv 0 0, 7 7 Chow NO 7 10mg/kg 15 43-45 3 NMRI/♀/ SEQ ID 1.13 iv 0 0, 7 7 Chow NO 8 mg/mlequimolar 11.3 mg/kg 17 49-51 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 7 7 Chow NO9 mg/ml equimolar 12.7 mg/kg 18 52-54 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 7 7Chow NO 10 mg/ml equimolar 12.7 mg/kg D 19 55-57 3 NMRI/♀/ Saline — iv 00, 7, 10 Chow 10 20 58-60 3 NMRI/♀/ SEQ ID 1 mg/ml iv 0 0, 7, 10 Chow NO7 10 10 mg/kg 21 61-63 3 NMRI/♀/ SEQ ID 1.13 iv 0 0, 7, 10 Chow NO 8mg/ml 10 equimolar 11.3 mg/kg 24 70-72 3 NMRI/♀/ SEQ ID 1.27 iv 0 0, 7,10 Chow NO 10 mg/ml 10 equimolar 12.7 mg/kg A 25 73-75 3 NMRI/♀/ Saline— iv 0 0, 1 1 Chow

Dose administration. NMRI female animals, app. 20 g at arrival, weredosed with 10 ml per kg BW (according to day 0 bodyweight) i.v. of thecompound formulated in saline or saline alone according to Table 5.

Sampling of liver and Kidney tissue. The animals were anaesthetized with70% CO₂-30% O₂ and sacrificed by cervical dislocation according to Table4. One half of the large liver lobe and one kidney were minced andsubmerged in RNAIater.

Total RNAwas extracted from maximum 10 mg of tissue homogenized bybead-milling in the presence of MagNA Pure LC RNA Isolation Tissuebuffer (Roche cat. no 03 604 721 001) using the MagNa Pure 96 CellularRNA Large Volume Kit (Roche cat no. 5467535001), according to themanufacturer's instructions. First strand synthesis was performed usingReverse Transcriptase reagents from Ambion according to themanufacturer's instructions. For each sample 0.5 μg total RNAwasadjusted to (10.8 μl) with RNase free H₂O and mixed with 2 μl randomdecamers (50 μM) and 4 μl dNTP mix (2.5 mM each dNTP) and heated to 70°C. for 3 min after which the samples were rapidly cooled on ice. 2 μl10× Buffer RT, 1 μl MMLV Reverse Transcriptase (100 U/μl) and 0.25 μlRNase inhibitor (10 U/μl) were added to each sample, followed byincubation at 42° C. for 60 min, heat inactivation of the enzyme at 95°C. for 10 min and then the sample was cooled to 4° C. cDNA samples werediluted 1:5 and subjected to RT-QPCR using Taqman Fast Universal PCRMaster Mix 2× (Applied Biosystems Cat #4364103) and Taqman geneexpression assay (mPCSK9, Mn00463738_m1 and mActin #4352341E) followingthe manufacturers protocol and processed in an Applied BiosystemsRT-qPCR instrument (7500/7900 or ViiA7) in fast mode.

Example 6. In Vitro Cleavage of Different DNA/PO-Unkers

FAM-labeled ASOs with different DNA/PO-linkers (PO linkers) weresubjected to in vitro cleavage either in S1 nuclease extract (FIG. 6A),Liver or kidney homogenates or Serum FAM-labeled ASOs 100 μM withdifferent DNA/PO-linkers were subjected to in vitro cleavage by S1nuclease in nuclease buffer (60 U pr. 100 μL) for 20 and 120 minutes(A).

The enzymatic activity was stopped by adding EDTA to the buffersolution. The solutions were then subjected to AIE HPLC analyses on aDionex Ultimate 3000 using an Dionex DNApac p-100 column and a gradientranging from 10 mM-1 M sodium perchlorate at pH 7.5. The content ofcleaved and non cleaved oligonucleotide were determinded against astandard using both a fluoresense detector at 615 nm and a uv detectorat 260 nm.

SEQ Linker % cleaved after 120 min ID NO sequence % cleaved after 20 minS1 S1 20 — 2 5 18 a 29.1 100 17 ca 40.8 100 16 tca 74.2 100 19 gac 22.9n.d

Conclusion: The PO linkers (or region B as referred to herein) resultsin the conjugate (or group C) being cleaved off, and both the lengthand/or the sequence composition of the linker can be used tomodulatesusceptibility to nucleolytic cleavage of region B. The Sequence ofDNA/PO-linkers can modulate the cleavage rate as seen after 20 min inNuclease S1 extract Sequence selection for region B (e.g. for theDNA/PO-linker) can therefore also be used to modulate the level ofcleavage in serum and in cells of target tissues.

Liver, kidney and Serum (B) were spiked with oligonucleotide SEQ ID NO16 to concentrations of 200 μg/g tissue. Liver and kidney samplescollected from NMRI mice were homogenized in a homogenisation buffer(0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjustedwith 1 N NaOH). The homogenates were incubated for 24 hours at 37° andthereafter the homogenates were extracted with phenol—chloroform. Thecontent of cleaved and non cleaved oligonucleotide in the extract fromliver and kidney and from the serum were determinded against a standardusing the above HPLC method.

% cleaved after % cleaved after % cleaved after Linker 24 hrs liver 24hrs kidney 24 hours in Seq ID Sequence homogenate homogenate serum 16tca 83 95 0

Conclusion: The PO linkers (or region B as referred to herein) resultsin cleavage of the conjugate (or group C) from the oligonucleotide, inliver or kidney homogenate, but not in serum.

Note: cleavage in the above assays refers to the cleavage of thecleavable linker, the oligomer or region A should remain functionallyintact. The susceptibility to cleavage in the above assays can be usedto determine whether a linker is biocleavable or physiologically labile.

Example 7a: In Vivo Inhibition of FVII (1 mg/kg)

An in vivo mouse study was prepared using a total of 6 groups of mice(n=3). Each mouse was administered a single i.v. dose of LNA compoundtargeting FVII mRNA, at 1 mg/kg or equimolar compared to SEQ ID #12. Asaline control group was included. The mice were pre-bled 1 day beforeadministration, and subsequent bleeds were taken at day 1 and 2 afteradministration. The mice were sacrificed at days 4, liver, kidney, andblood samples were taken. See table 7 for study setup.

Factor VII serum levels, mRNA levels, and oligonucleotide tissue contentwere measured using standard assay techniques.

Conclusions: The DNA/PO-linker (PO) improves the FVII protein downregulation in serum (FIG. 18) for FVII mRNA targeting LNAoligonucleotides with cholesterol conjugates when comparing to thewidely used dithio linker (disulphide) (PO linker SEQ ID #15 compare toSS linker SEQ ID #14). Using GalNAc as conjugate it is apparent that thePO linker improves the down regulation of FVII protein when compared tothe aminolinked conjugated LNA oligonucleotide (PO linker SEQ ID #13compare to amino linked SEQ ID #12). The GalNac conjugate is known to bebiocleavable (possibly due to the peptide linker), and as such itappears that the PO linker further enhances the release of active andpotent compound in the target cell. These data corresponds to the mRNAexpression data (FIG. 19). The tissue content of oligonucleotide inkidney and liver shows how the conjugates change the distribution (FIG.20). It is seen that the two cholesterol conjugated compounds givessimilar distribution (compare SEQ ID #14 and #15) so with the enhancedmRNA and FVII protein down regulation of the PO linker compound (SEQ ID#15) it is seen how the PO linker enhances the activity of the FVIItargeting LNA oligonucleotide when comparing to SEQ ID #14.

Materials and Methods:

Experimental Design:

TABLE 7 termination dose time point group (d0) group compound post dosesize mg/kg 1 saline d4 3 none 2 SEQ ID #11 d4 3 1 3 SEQ ID #12 d4 3 1 4SEQ ID #13 d4 3 1 5 SEQ ID #14 d4 3 1 6 SEQ ID #15 d4 3 1

Female mice were administered iv and liver, kidney, and blood weresampled at sacrifice on day 4, Additional blood draws were made beforedosing and also on day 1 and 2 after dosing.

Example 7b: In Vivo Inhibition of FVII (0.1 and 0.25 mg/kg)

An in vivo mouse study was prepared using a total of 7 groups of mice(n=3). Each mouse was administered a single i.v. dose of LNA compoundtargeting FVII mRNA, at either 0.1 mg/kg or 0.25 mg/kg in equimolaramount compared to SEQ ID #12. A saline control group was included. Themice were pre-bled 1 day before administration, and subsequent bleedswere taken at days 4, 7, 11, 14, and 18 after administration. The micewere sacrificed at days 24, liver, kidney, and blood samples were taken.See table 8 for study setup. Factor VII serum levels, mRNA levels, andoligonucleotide tissue content were measured using standard assaytechniques

Conclusions: The DNA/PO-linker (PO) improves the FVII protein downregulation (FIG. 21) for FVII mRNA targeting LNA oligonucleotides withcholesterol conjugates when comparing to the widely used dithio linker(PO linker SEQ ID #15 compare to SS linker SEQ ID #14) at both 0.1 mg/kgand 0.25 mg/kg. Using GalNAc as conjugate mRNA data (FIG. 22) suggestthat the PO linker improves the down regulation when compared to theaminolinked conjugated LNA oligonucleotide (PO linker SEQ ID #13 compareto amino linked SEQ ID #12). The mRNA expression data (FIG. 22) supportsthe improved activity of the PO linker compound (SEQ ID #15) compared tothe dithio linked conjugate (SEQ ID #14). The tissue content ofoligonucleotide in kidney and liver shows how the conjugates change thedistribution (FIG. 23). Data suggest that the PO linker enhances theuptake both in liver and kidney at these dose ranges for bothCholesterol conjugate and GalNAc conjugate (compare SEQ ID #14 and #15and compare SEQ ID #13 to #12)

Materials and Methods:

Experimental Design:

TABLE 8 termination dose time point group (d0) group compound post dosesize mg/kg 1 Saline d24 3 none 2 SEQ ID #12 d24 3 0.1 3 SEQ ID #13 d24 30.1 4 SEQ ID #14 d24 3 0.1 5 SEQ ID #14 d24 3 0.25 6 SEQ ID #15 d24 30.1 7 SEQ ID #15 d24 3 0.25

Male mice were administered iv and liver, kidney, and blood were sampledat sacrifice on day 24, Additional blood draws were made before dosingand also on day 4, 7, 11, 14, and 18 after dosing.

Example 8. In Vivo Silencing of ApoB mRNA with Different Conjugates andPO-Linker

To explore the impact of the biocleavable DNA/PO-linker on additionalconjugates C57BL6I mice were treated i.v. with saline control or with asingle dose of 1 mg/kg kg for parent compound #1 or equimolarly of ASOconjugated to Mono-GalNAc, Folic acid, Fam or Tocopherol, either withoutbiocleavable linker, with Dithio-linker (SS) or with DNA/PO-linker (PO).After 7 days animals were sacrificed and RNA was isolated from liver andkidney samples and analyzed for ApoB mRNA expression (FIG. 24)

Conclusions:

For all 4 conjugates the DNA/PO-linker improves ApoB knock down in theliver compared to the widely used dithio-linker (compare #27 with #26,#30 with #29, #33 with #32 and #36 with #35). For mono-GalNAc andTocopherol the DNA/PO-linker improves knock down of ApoB in the livereven compared to unconjugated compound (compare #30 and #36 with #1).Tocopherol combined with a DNA/PO-linker shows capability of redirectinga compound from kidney to liver (compare A and B, #36 with #1)

Materials and Methods:

Experimental Design;

TABLE 9 Com- Animal pound strain/ Seq ID, Sacri- Gr. Animal gender/ doseAdm. Dosing fice no. ID no. feed 1 mg/kg Route Day Day 1  1-5  NMRI ♀- 1i.v. 0 7 Chow 2  5-10 NMRI ♀- 28 i.v. 0 7 Chow 3 11-15 NMRI ♀- 29 i.v. 07 Chow 4 16-20 NMRI ♀- 30 i.v.. 0 7 Chow 5 21-25 NMRI ♀- 25 i.v. 0 7Chow 6 26-30 NMRI ♀- 26 i.v. 0 7 Chow 7 31-35 NMRI ♀- 27 i.v. 0 7 Chow 836-40 NMRI ♀- NaCl i.v. 0 7 Chow 0.9% 1 1-5 NMRI ♀- 1 i.v. 0 7 Chow 2 5-10 NMRI ♀- 31 i.v. 0 7 Chow 3 11-15 NMRI ♀- 32 i.v. 0 7 Chow 4 16-20NMRI ♀- 33 i.v.. 0 7 Chow 5 21-25 NMRI ♀- 34 i.v. 0 7 Chow 6 26-30 NMRI♀- 35 i.v. 0 7 Chow 7 31-35 NMRI ♀- 36 i.v. 0 7 Chow 8 36-40 NMRI ♀-NaCl i.v. 0 7 Chow 0.9%

Dose administration and sampling. C57BL6 mice were dosed i.v. with asingle dose of 1 mg/kg ASO formulated in saline or saline aloneaccording to the above table. Animals were sacrificed at day 7 afterdosing and liver and kidney were sampled.

RNA isolation and mRNA analysis. Total RNA was extracted from liver andkidney samples and ApoB mRNA levels were analyzed using a branched DNAassay.

Example 9. In Vitro Silencing of Target X mRNA with Looped LNA ASO withPO-Linker

Blocker groups might be beneficial regarding tolerability, specificityor reduced off-target effect of ASOs but challenging in terms ofpreserving the activity of the original, unblocked ASO. As an examplefor a blocker group we used a complementary sequence which is connectedto the oligonucleotide by a non-complementary nucleotide stretchgenerating a hairpin loop. The unpaired bases in the loop where either 3DNA nucleotides with Phophodiester-backbone (PO-linker) or the same DNAnucleotides with Phosphorothioate-backbone. To test the activity of thelooped LNA-ASOs Neuro 2a cells were treated with 1 μM ASO in a gymnosisassay and RNA was extracted and subjected to RT-QPCR to analyze fortarget X mRNA knock down (FIG. 25).

Conclusion: Looped ASOs with PO-linker (Seq ID #21 and #23) showedimproved target X mRNA knock down compared to the same ASO sequencewithout PO-linker (Seq ID #22 and #24).

Materials and Methods:

Gymnosis Assay in N2a Cells:

Neuro 2a (mouse neuroblastoma) cells were seeded in 24 well plates with1.8×10⁴ cells/well and treated with 1 μM looped LNA ASOs with andwithout PO-linker, respectively in DMEM+Glutamax (gibco-life,#61965-026), 2 mM Glutamine, 10% FBS, 1 mM Sodium Pyruvate, 25 μg/mlGentamicin.

Total RNA Isolation and First strand synthesis. Total RNA was extractedafter 6 days gymnosis using the Qiagen RNeasy kit (Qiagen cat. no.74106) according to the manufacturer's instructions. First strandsynthesis was performed using Reverse Transcriptase reagents from Ambionaccording to the manufacturer's instructions.

For each sample 0.5 μg total RNA was mixed with 2 μl random decamers (50μM) and 4 μl dNTP mix (2.5 mM each dNTP) and heated to 70° C. for 3 minafter which the samples were rapidly cooled on ice. 2 μl 10× Buffer RT,1 μl MMLV Reverse Transcriptase (100 U/μl) and 0.25 μl RNase inhibitor(10 U/μl) were added to each sample, followed by incubation at 42° C.for 60 min, heat inactivation of the enzyme at 95° C. for 10 min andthen the sample was cooled to 4° C. cDNA samples were diluted 1:5 andsubjected to RT-QPCR using Taqman Fast Universal PCR Master Mix 2×(Applied Biosystems Cat #4364103) and Taqman gene expression assayagainst target X following the manufacturers protocol and processed inan Applied Biosystems RT-qPCR instrument (ViiA7) in fast mode. Target XmRNA expression was normalized to Beta actin mRNA expression (mBACT#4352341E) and compared to mock mRNA levels.

Example 10: Non-Human Primate Study

The primary objective for this study is to investigate selected lipidmarkers over 7 weeks after a single slow bolus injection of anti-PCSK9and anti-ApoB LNA conjugated compounds to cynomolgus monkeys and assessthe potential toxicity of compounds in monkey. The compounds used inthis study are SEQ ID NOs 46 and 49, 5 and 54, which were prepared insterile saline (0.9%) at an initial concentration of 0.625 and 2.5mg/ml).

Male (PCSK9) or female monkeys (ApoB) monkeys of at least 24 months oldare used, and given free access to tap water and 180 g of MWM(E) SQCSHORT expanded diet (Dietex France, SDS, Saint Gratien, France) will bedistributed daily per animal. The total quantity of food distributed ineach cage will be calculated according to the number of animals in thecage on that day. In addition, fruit or vegetables will be given dailyto each animal. The animals will be acclimated to the study conditionsfor a period of at least 14 days before the beginning of the treatmentperiod. During this period, pre-treatment investigations will beperformed. The animals are dosed i.v. at a dose if, for example, 0.25mg/kg or 1 mg/kg. The dose volume will be 0.4 mL/kg. 2 animals are usedper group. After three weeks, the data will be analyzed and a secondgroup of animals using a higher or lower dosing regimen may beinitiated—preliminary dose setting is 0.5 mg/kg and 1 mg/kg, or lowerthan that based on the first data set.

The dose formulations will be administered once on Day 1. Animals willbe observed for a period of 7 weeks following treatment, and will bereleased from the study on Day 51. Day 1 corresponds to the first day ofthe treatment period. Clinical observations and body weight and foodintake (per group) will be recorded prior to and during the study.

Blood is sampled and analysis at the following time points:

Study Day Parameters −8 RCP, L, Apo-B, PCSK9*, OA −1 L, Apo-B, PCSK9*,PK, OA 1 Dosing 4 LSB, L, Apo-B, PCSK9*, OA 8 LSB, L, Apo-B, PCSK9*, PK,OA 15 RCP, L, Apo-B, PCSK9* PK, OA 22 LSB, L, Apo-B, PCSK9* PK, OA 29 L,Apo-B, PCSK9* PK, OA 36 LSB, L, Apo-B, PCSK9* PK, OA 43 L, PK, Apo-B,PCSK9* PK, OA 50 RCP, L, Apo-B, PCSK9* PK, OA RCP 0 routine clinicalpathology, LSB = liver safety biochemistry, PK = pharmacokinetics, OA =other analysis, L = Lipids.

Blood Biochemistry

The following parameters will be determined for all surviving animals atthe occasions indicated below:

-   -   full biochemistry panel (complete list below)—on Days −8, 15 and        50,    -   liver Safety (ASAT, ALP, ALAT, TBIL and GGT only)—on Days 4, 8,        22 and 36,    -   lipid profile (Total cholesterol, HDL-C, LDL-C and        Triglycerides) and Apo-B only—on Days −1, 4, 8, 22, 29, 36, and        43.

Blood (approximately 1.0 mL) is taken into lithium heparin tubes (usingthe ADVIA 1650 blood biochemistry analyzer): Apo-B, sodium, potassium,chloride, calcium, inorganic phosphorus, glucose, HDL-C, LDL-C, urea,creatinine, total bilirubin (TBIL), total cholesterol, triglycerides,alkaline phosphatase (ALP), alanine aminotransferase (ALAT), aspartateaminotransferase (ASAT), creatine kinase, gamma-glutamyl transferase(GGT), lactate dehydrogenase, total protein, albumin, albumin/globulinratio.

Analysis of blood: Blood samples for PCSK9 analysis will be collectedfrom Group 16 animals only on Days −8, −1, 4, 8, 15, 22, 29, 36, 43 and50.

Venous blood (approximately 2 mL) will be collected from an appropriatevein in each animal into a Serum Separating Tube (SST) and allowed toclot for at least 60±30 minutes at room temperature. Blood will becentrifuged at 1000 g for 10 minutes under refrigerated conditions (setto maintain +4° C.). The serum will be transferred into 3 individualtubes and stored at −80° C. until analyzed at CitoxLAB France using anELISA method (Circulex Human PCSK9 ELISA kit, CY-8079, validated forsamples from cynomolgus monkey).

Other Analysis: WO2011009697 & WO2010142805 provides the methods for thefollowing analysis: qPCR, PCSK9/ApoB mRNA analysis, Other analysisincludes PCSK9/ApoB protein ELISA, serum Lp(a) analysis with ELISA(Mercodia No. 10-1106-01), tissue and plasma oligonucleotide analysis(drug content), Extraction of samples, standard—and QC-samples,Oligonucleotide content determination by ELISA.

Example 11: Liver and Kidney Toxicity Assessment in Rat

Compounds of the invention can be evaluated for their toxicity profilein rodents, such as in mice or rats. By way of example the followingprotocol may be used: Wistar Han Crl:WI(Han) are used at an age ofapproximately 8 weeks old. At this age, the males should weighapproximately 250 g. All animals have free access to SSNIFF R/M-Hpelleted maintenance diet (SSNIFF Spezialdiaten GmbH, Soest, Germany)and to tap water (filtered with a 0.22 μm filter) contained in bottles.The dose level of 10 and 40 mg/kg/dose is used (sub-cutaneousadministration) and dosed on days 1 and 8. The animals are euthanized onDay 15. Urine and blood samples are collected on day 7 and 14. Aclinical pathology assessment is made on day 14. Body weight isdetermined prior to the study, on the first day of administration, and 1week prior to necropsy. Food consumption per group will be assesseddaily. Blood samples are taken via the tail vein after 6 hours offasting. The following blood serum analysis is performed: erythrocytecount mean cell volume packed cell volume hemoglobin mean cellhemoglobin concentration mean cell hemoglobin thrombocyte countleucocyte count differential white cell count with cell morphologyreticulocyte count, sodium potassium chloride calcium inorganicphosphorus glucose urea creatinine total bilirubin total cholesteroltriglycerides alkaline phosphatase alanine aminotransferase aspartateaminotransferase total protein albumin albumin/globulin ratio.Urinalysis are performed α-GST, β-2 Microglobulin, Calbindin, Clusterin,Cystatin C, KIM-1, Osteopontin, TIMP-1, VEGF. and NGAL. Seven analytes(Calbindin, Clusterin, GST-α, KIM-1, Osteopontin, TIMP-1, VEGF) will bequantified under Panel 1 (MILLIPLEX® MAP Rat Kidney Toxicity MagneticBead Panel 1, RKTX1MAG-37K). Three analytes (β-2 Microglobulin, CystatinC, Lipocalin-2/NGAL) will be quantified under Panel 2 (MILLIPLEX® MAPRat Kidney Toxicity Magnetic Bead Panel 2, RKTX2MAG-37K). The assay forthe determination of these biomarkers' concentration in rat urines isbased on the Luminex xMAP® technology. Microspheres coated withanti-α-GST/β-2 microglobulin/calbindin/clusterin/cystacinC/KIM-1/osteopontin/TIMP-1/VEGF/NGAL antibodies are color-coded with twodifferent fluorescent dyes. The following parameters are determined(Urine using the ADVIA 1650): Urine protein, urine creatinine.Quantitative parameters: volume, pH (using 10-Multistix SG teststrips/Clinitek 500 urine analyzer), specific gravity (using arefractometer). Semi-quantitative parameters (using 10-Multistix SG teststrips/Clinitek 500 urine analyzer): proteins, glucose, ketones,bilirubin, nitrites, blood, urobilinogen, cytology of sediment (bymicroscopic examination). Qualitative parameters: Appearance, color.After sacrifice, the body weight and kidney, liver and spleen weight aredetermined and organ to body weight ratio calculated. Kidney and liversamples will be taken and either frozen or stored in formalin.Microscopic analysis is performed.

1.-52. (canceled)
 53. A method of synthesizing an oligomeric compound8-35 nucleotides in length, comprising three regions: a first region(region A), which is an antisense oligomer comprising 7-26 contiguousnucleotides complementary to a nucleic acid target, wherein the firstregion comprises at least one nucleoside analog and wherein theinternucleoside linkages of the first region comprise at least 50%internucleoside linkages other than phosphodiester; a second region(region B) which comprises between 1-10 nucleotides, which is covalentlylinked to the 5′ or 3′ nucleotide of the first region by aninternucleoside linkage, wherein either: (i) the internucleoside linkagebetween the first and second region is a phosphodiester linkage and thenucleoside of the second region adjacent to the first region is eitherDNA or RNA; and/or (ii) at least 2 nucleosides of the second region arephosphodiester linked DNA or RNA nucleosides; a third region whichcomprises a conjugate moiety, a targeting moiety, or a blocking moiety,wherein the third region is covalently linked to the second region, saidmethod comprising either: (a) a step of providing an oligonucleotidesynthesis support to which is attached a moiety selected from the groupconsisting of: (i) a linker group (—Y—), (ii) a group (X—) selected fromthe group consisting of a conjugate, a targeting group and a blockinggroup, and (iii) a —Y—X group; and (b) a step of oligonucleotidesynthesis of region B followed by region A, or: (a′) a step ofoligonucleotide synthesis of a first region (A) and a second region (B),wherein the first region (A) is attached to an oligonucleotide synthesissupport and the synthesis step is followed by (b′) a step of adding amoiety selected from the group consisting of: (i) a linker group (—Y —);(ii) a group (X—) selected from the group consisting of a conjugate, atargeting group or a blocking group, and (iii) an —Y—X group; followedby (c) the cleavage of the oligomeric compound from the support,wherein, if the moiety is a linker group (Y), said method furthercomprises, (d) a further step of adding a conjugate, a blocking, ortargeting group to the linker group (Y), wherein said step is performedeither prior to or subsequent to cleavage of the oligomeric compoundfrom the oligonucleotide synthesis support.
 54. The method of claim 53,comprising steps (a), (b) and (c).
 55. The method of claim 53,comprising steps (a′), (b′) and (e)
 56. A method of synthesizing anoligomeric compound of 8-35 nucleotides in length, comprising threeregions: a first region (region A), which is an antisense oligomer of7-26 contiguous nucleotides complementary to a nucleic acid target,wherein the first region comprises at least four nucleoside analoguesand wherein the internucleoside linkages of the first region areinternucleoside linkages other than phosphodiester; a second region(region B) which is covalently linked to the 5′ or 3′ nucleotide of thefirst region via a phosphodiester internucleoside linkage, wherein thesecond region consists of 2-10 phosphodiester linked DNA or RNAnucleosides; and a third region which comprises a conjugate moiety or atargeting moiety wherein the third region is covalently linked to thesecond region, said method comprising either: (a) a step of providing anoligonucleotide synthesis support to which is attached a moiety selectedfrom the group consisting of: (i) a linker group (—Y—), (ii) a group(X—) selected from the group consisting of a conjugate and a targetinggroup, and (iii) a —Y—X group; and (b) a step of oligonucleotidesynthesis of region B followed by region A, or: (a′) a step ofoligonucleotide synthesis of a first region (A) and a second region (B),wherein the first region (A) is attached to an oligonucleotide synthesissupport and the synthesis step is followed by (b′) a step of adding amoiety selected from the group consisting of: (i) a linker group (—Y—);(ii) a group (X—) selected from the group consisting of a conjugate anda targeting group, and (iii) a —Y—X group, followed by (c) the cleavageof the oligomeric compound from the support, wherein, if the moiety is alinker group (Y), said method further comprises (d) a further step ofadding a conjugate or a targeting group to the linker group (Y), whereinsaid further step is performed either prior to or subsequent to cleavageof the oligomeric compound from the oligonucleotide synthesis support.57. The method of claim 56, comprising steps (a), (b) and (e).
 58. Themethod of claim 56, comprising steps (a′), (b′) and (e).
 59. The methodaccording to claim 56, wherein the internucleoside linkages other thanphosphodiester in the first region of the oligomeric compound areselected from the group consisting of phosphorothioate linkages,phosphorodithioate linkages and boranophosphate linkages.
 60. The methodaccording to claim 56, wherein the first region is a gapmer, a mixmer,or a totalmer.
 61. The method according to claim 56, wherein the firstregion comprises at least one bicyclic nucleotide analogue (LNA). 62.The method according to claim 56, wherein the second region iscovalently linked to the third region at the terminal nucleoside of thesecond region.
 63. The method according to claim 56, wherein the secondand third regions are covalently joined by a linker group.
 64. Themethod according to claim 56, wherein the linkage between the second andthird regions comprises a group selected from phosphodiester, aphosphorothioate, a phosphorodithioate and a boranophosphate group. 65.The method according to claim 56, wherein the third region comprises anon-nucleotide moiety selected from a sterol and a carbohydrate.
 66. Themethod according to claim 13, wherein the third region comprisescholesterol.
 67. The method according to claim 13, wherein the thirdregion comprises a GalNac cluster.
 68. The method according to claim 56,wherein the third region comprises a moiety selected from the groupconsisting of: a lipophilic group, a protein, a peptide, an antibody orfragment thereof, a polymer, a reporter group, a dye, a receptor ligand,a small molecule drug, a prodrug, and a vitamin.
 69. A method ofsynthesizing an oligomeric compound composed of three regions: a firstregion (region A), which is an antisense oligomer of 10-22 contiguousnucleotides complementary to a nucleic acid target, wherein the firstregion is a gapmer; a second region (region B) which comprises between1-10 nucleotides, which is covalently linked to the 5′ or 3′ nucleotideof the first region, wherein: (i) the internucleoside linkage betweenthe first and second region is a phosphodiester linkage and thenucleoside of the second region adjacent to the first region is either aDNA nucleotide or an RNA nucleotide; and/or (ii) at least one nucleotideof the second region is a phosphodiester linked DNA nucleotide or RNAnucleotide; and a third region which comprises a conjugate moiety or atargeting moiety, wherein the third region is covalent linked to the 5′or 3′ terminus of the second region, said method comprising either: (a)a step of providing an oligonucleotide synthesis support to which isattached a moiety selected from the group consisting of: (i) a linkergroup (—Y—); (ii) a group (X—) selected from the group consisting of aconjugate and a targeting group, and (iii) a —Y—X group and (b) a stepof oligonucleotide synthesis of region B followed by region A, or: (a′)a step of oligonucleotide synthesis of a first region (A) and a secondregion (B), wherein the first region (A) is attached to anoligonucleotide synthesis support and the synthesis step is followed by(b′) a step of adding a moiety selected from the group consisting of:(i) a linker group (—Y—); (ii) a group (X—) selected from the groupconsisting of a conjugate and a targeting group, and (iii) a —Y—X group,followed by (c) the cleavage of the oligomeric compound from the supportwherein, if the moiety is a linker group (Y), said method furthercomprises (d) a further step of adding a conjugate or a targeting groupto the linker group (Y), wherein said further step is performed eitherprior to or subsequent to cleavage of the oligomeric compound from theoligonucleotide synthesis support.
 70. The method of claim 69,comprising steps (a), (b) and (c).
 71. The method of claim 69,comprising steps (a′), (b′) and (c).
 72. The method according to claim69, wherein the internucleoside linkages of the first region comprise atleast one internucleoside linkage other than phosphodiester.
 73. Themethod according to claim 72, wherein the internucleoside linkages otherthan phosphodiester in the first region of the oligomeric compound areselected from the group consisting of phosphorothioate,phosphorodithioate and boranophosphate.
 74. The method according toclaim 73, wherein at least 75%, of the internucleoside linkages of thefirst region are other than phosphodiester.
 75. The method according toclaim 73, wherein all of the internucleoside linkages of the firstregion are other than phosphodiester.
 76. The method according to claim69, wherein the first region of the oligomeric compound comprises atleast one bicyclic nucleotide analogue (LNA).
 77. The method of claim68, wherein the first region of the oligomeric compound is covalentlylinked to the second region via a phosphodiester linkage.
 78. The methodaccording to claim 69, wherein the second region of the oligomericcompound is 1 to 4 nucleotides long.
 79. The method according to claim69, wherein the second region consists of one DNA nucleotide or one RNAnucleotide.
 80. The method according to claim 69, wherein the secondregion of the oligomeric compound comprises at least two nucleotidesselected from the group of DNA nucleotides and RNA nucleotides, whereinthe nucleotides are linked by phosphodiester linkages.
 81. The methodaccording to claim 69, wherein the second region of the oligomericcompound comprises at least two nucleotides and the linkage between thefirst two nucleotide adjacent to the first region is a phosphodiesterlinkage.
 82. The method according to claim 69, wherein the second regionof the oligomeric compound is covalently linked to the third region atthe terminal nucleoside of the second region.
 83. The method accordingto claim 81, wherein the third region of the oligomeric compound iscovalently linked to the second region via a phosphodiester linkage. 84.The method according to claim 69, wherein the first region and thesecond region of the oligomeric compound are covalently linked by aphosphodiester linkage and the second region and the third region arecovalently linked by a phosphodiester linkage.
 85. The method accordingto claim 69, wherein the third region of the oligomeric compoundcomprises cholesterol.
 86. The method according to claim 69, wherein thethird region of the oligomeric compound comprises a GalNac cluster.